Microtubule minus end motors kinesin-14 and dynein drive nuclear congression in parallel pathways.

PubMed

Scheffler, Kathleen; Minnes, Refael; Fraisier, Vincent; Paoletti, Anne; Tran, Phong T

2015-04-13

Microtubules (MTs) and associated motors play a central role in nuclear migration, which is crucial for diverse biological functions including cell division, polarity, and sexual reproduction. In this paper, we report a dual mechanism underlying nuclear congression during fission yeast karyogamy upon mating of haploid cells. Using microfluidic chambers for long-term imaging, we captured the precise timing of nuclear congression and identified two minus end-directed motors operating in parallel in this process. Kinesin-14 Klp2 associated with MTs may cross-link and slide antiparallel MTs emanating from the two nuclei, whereas dynein accumulating at spindle pole bodies (SPBs) may pull MTs nucleated from the opposite SPB. Klp2-dependent nuclear congression proceeds at constant speed, whereas dynein accumulation results in an increase of nuclear velocity over time. Surprisingly, the light intermediate chain Dli1, but not dynactin, is required for this previously unknown function of dynein. We conclude that efficient nuclear congression depends on the cooperation of two minus end-directed motors. © 2015 Scheffler et al.

Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

PubMed

Siglin, Amanda E; Sun, Shangjin; Moore, Jeffrey K; Tan, Sarah; Poenie, Martin; Lear, James D; Polenova, Tatyana; Cooper, John A; Williams, John C

2013-01-01

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

Dynein-ADP as a force-generating intermediate revealed by a rapid reactivation of flagellar axoneme.

PubMed Central

Tani, T; Kamimura, S

1999-01-01

Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration ( approximately 80 microM) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate. PMID:10465762

A mouse neurodegenerative dynein heavy chain mutation alters dynein motility and localization in Neurospora crassa.

PubMed

Sivagurunathan, Senthilkumar; Schnittker, Robert R; Nandini, Swaran; Plamann, Michael D; King, Stephen J

2012-09-01

Cytoplasmic dynein is responsible for the transport and delivery of cargoes in organisms ranging from humans to fungi. Dysfunction of dynein motor machinery due to mutations in dynein or its activating complex dynactin can result in one of several neurological diseases in mammals. The mouse Legs at odd angles (Loa) mutation in the tail domain of the dynein heavy chain has been shown to lead to progressive neurodegeneration in mice. The mechanism by which the Loa mutation affects dynein function is just beginning to be understood. In this work, we generated the dynein tail mutation observed in Loa mice into the Neurospora crassa genome and utilized cell biological and complementing biochemical approaches to characterize how that tail mutation affected dynein function. We determined that the Loa mutation exhibits several subtle defects upon dynein function in N. crassa that were not seen in mice, including alterations in dynein localization, impaired velocity of vesicle transport, and in the biochemical properties of purified motors. Our work provides new information on the role of the tail domain on dynein function and points out areas of future research that will be of interest to pursue in mammalian systems. 2012 Wiley Periodicals, Inc

A Mouse Neurodegenerative Dynein Heavy Chain Mutation Alters Dynein Motility and Localization in Neurospora crassa

PubMed Central

Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

2013-01-01

Cytoplasmic dynein is responsible for the transport and delivery of cargoes in organisms ranging from humans to fungi. Dysfunction of dynein motor machinery due to mutations in dynein or its activating complex dynactin can result in one of several neurological diseases in mammals. The mouse Legs at odd angles (Loa) mutation in the tail domain of the dynein heavy chain has been shown to lead to progressive neurodegeneration in mice. The mechanism by which the Loa mutation affects dynein function is just beginning to be understood. In this work, we generated the dynein tail mutation observed in Loa mice into the Neurospora crassa genome and utilized cell biological and complementing biochemical approaches to characterize how that tail mutation affected dynein function. We determined that the Loa mutation exhibits several subtle defects upon dynein function in N. crassa that were not seen in mice, including alterations in dynein localization, impaired velocity of vesicle transport, and in the biochemical properties of purified motors. Our work provides new information on the role of the tail domain on dynein function and points out areas of future research that will be of interest to pursue in mammalian systems. PMID:22991199

The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

PubMed Central

Myster, S H; Knott, J A; O'Toole, E; Porter, M E

1997-01-01

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex. Images PMID:9247642

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

PubMed

Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

2000-01-01

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast

PubMed Central

Yamamoto, Ayumu; West, Robert R.; McIntosh, J. Richard; Hiraoka, Yasushi

1999-01-01

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination. PMID:10366596

Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1

PubMed Central

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C.; Aviram, Micha; Parvari, Ruti

2011-01-01

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. PMID:21496787

JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

PubMed Central

Drerup, Catherine M.; Nechiporuk, Alex V.

2013-01-01

Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants (jip3nl7) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3nl7, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3nl7, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein. PMID:23468645

Loss-of-Function Mutations in a Human Gene Related to Chlamydomonas reinhardtii Dynein IC78 Result in Primary Ciliary Dyskinesia

PubMed Central

Pennarun, Gaëlle; Escudier, Estelle; Chapelin, Catherine; Bridoux, Anne-Marie; Cacheux, Valère; Roger, Gilles; Clément, Annick; Goossens, Michel; Amselem, Serge; Duriez, Bénédicte

1999-01-01

Summary Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects. PMID:10577904

Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1.

PubMed

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C; Aviram, Micha; Parvari, Ruti

2011-05-13

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Clinical and Genetic Aspects of Primary Ciliary Dyskinesia / Kartagener Syndrome

PubMed Central

Leigh, Margaret W.; Pittman, Jessica E.; Carson, Johnny L.; Ferkol, Thomas W.; Dell, Sharon D.; Davis, Stephanie D.; Knowles, Michael R.; Zariwala, Maimoona A.

2013-01-01

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (DNAH5) or intermediate (DNAI1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for PCD is available for the most common mutations. The respiratory manifestations of PCD (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of PCD patients have laterality defects (including situs inversus totalis and, less commonly, heterotaxy and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most PCD patients have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with PCD. PMID:19606528

An outer arm dynein light chain acts in a conformational switch for flagellar motility

PubMed Central

Patel-King, Ramila S.

2009-01-01

A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity. PMID:19620633

Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid.

PubMed

Lin, S X; Ferro, K L; Collins, C A

1994-11-01

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.

The Novel Zinc Finger Protein dASCIZ Regulates Mitosis in Drosophila via an Essential Role in Dynein Light-Chain Expression

PubMed Central

Zaytseva, Olga; Tenis, Nora; Mitchell, Naomi; Kanno, Shin-ichiro; Yasui, Akira; Heierhorst, Jörg; Quinn, Leonie M

2014-01-01

The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. Here we report the identification of the Drosophila ortholog of ASCIZ (dASCIZ) and show that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, we show that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp–LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. We speculate that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, our data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light-chain levels and a novel regulator of mitosis in flies. PMID:24336747

A Motor-Driven Mechanism for Cell-Length Sensing

PubMed Central

Rishal, Ida; Kam, Naaman; Perry, Rotem Ben-Tov; Shinder, Vera; Fisher, Elizabeth M.C.; Schiavo, Giampietro; Fainzilber, Mike

2012-01-01

Summary Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing. Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development. Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1. Thus, molecular motors critically influence cell-length sensing and growth control. PMID:22773964

A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein

PubMed Central

Gleave, Emma S.; Schmidt, Helgo; Carter, Andrew P.

2014-01-01

Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomycescerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins. PMID:24680784

Functional Architecture of the Outer Arm Dynein Conformational Switch*

PubMed Central

King, Stephen M.; Patel-King, Ramila S.

2012-01-01

Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia. PMID:22157010

TCTEX1D4 Interactome in Human Testis: Unraveling the Function of Dynein Light Chain in Spermatozoa

PubMed Central

Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; da Cruz e Silva, Edgar

2014-01-01

Abstract Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function. PMID:24606217

A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein.

PubMed

Gleave, Emma S; Schmidt, Helgo; Carter, Andrew P

2014-06-01

Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomycescerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

PubMed Central

Rompolas, Panteleimon; Patel-King, Ramila S.; King, Stephen M.

2012-01-01

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters. PMID:22855525

Quantitative Analysis of Pac1/LIS1-mediated Dynein Targeting: Implications for Regulation of Dynein Activity in Budding Yeast

PubMed Central

Markus, Steven M.; Plevock, Karen M.; St. Germain, Bryan J.; Punch, Jesse J.; Meaden, Christopher W.; Lee, Wei-Lih

2011-01-01

LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik1 recruits approximately equal amounts of Pac1 and Dyn1. Overexpression of Pac1 enhanced plus end targeting of Dyn1 and vice versa, while affinity-purification of Dyn1 revealed that it exists in a complex with Pac1 in the absence of Bik1, leading us to conclude that the Pac1-Dyn1 complex preassembles in the cytoplasm prior to loading onto Bik1-decorated plus ends. Strikingly, we found that Pac1-overexpression augments cortical dynein activity through a mechanism distinct from loss of She1, a negative regulator of dynein-dynactin association. While Pac1-overexpression enhances the frequency of cortical targeting for dynein and dynactin, the stoichiometry of these complexes remains relatively unchanged at the plus ends compared to that in wild-type cells (~3 dynein to 1 dynactin). Loss of She1, however, enhances dynein-dynactin association at the plus ends and the cell cortex, resulting in an apparent 1:1 stoichiometry. Our results reveal differential regulation of cortical dynein activity by She1 and Pac1, and provide a potentially new regulatory step in the off-loading model for dynein function. PMID:21294277

Clustering of Nuclei in Multinucleated Hyphae Is Prevented by Dynein-Driven Bidirectional Nuclear Movements and Microtubule Growth Control in Ashbya gossypii ▿ †

PubMed Central

Grava, Sandrine; Keller, Miyako; Voegeli, Sylvia; Seger, Shanon; Lang, Claudia; Philippsen, Peter

2011-01-01

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein. PMID:21642510

Clustering of nuclei in multinucleated hyphae is prevented by dynein-driven bidirectional nuclear movements and microtubule growth control in Ashbya gossypii.

PubMed

Grava, Sandrine; Keller, Miyako; Voegeli, Sylvia; Seger, Shanon; Lang, Claudia; Philippsen, Peter

2011-07-01

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.

The Dynein Gene Family in Chlamydomonas Reinhardtii

PubMed Central

Porter, M. E.; Knott, J. A.; Myster, S. H.; Farlow, S. J.

1996-01-01

To correlate dynein heavy chain (Dhc) genes with flagellar mutations and gain insight into the function of specific dynein isoforms, we placed eight members of the Dhc gene family on the genetic map of Chlamydomonas. Using a PCR-based strategy, we cloned 11 Dhc genes from Chlamydomonas. Comparisons with other Dhc genes indicate that two clones correspond to genes encoding the alpha and beta heavy chains of the outer dynein arm. Alignment of the predicted amino acid sequences spanning the nucleotide binding site indicates that the remaining nine clones can be subdivided into three groups that are likely to include representatives of the inner-arm Dhc isoforms. Gene-specific probes reveal that each clone represents a single-copy gene that is expressed as a transcript of the appropriate size (>13 kb) sufficient to encode a high molecular weight Dhc polypeptide. The expression of all nine genes is upregulated in response to deflagellation, suggesting a role in axoneme assembly or motility. Restriction fragment length polymorphisms between divergent C. reinhardtii strains have been used to place each Dhc gene on the genetic map of Chlamydomonas. These studies lay the groundwork for correlating defects in different Dhc genes with specific flagellar mutations. PMID:8889521

ZMYND10 stabilizes intermediate chain proteins in the cytoplasmic pre-assembly of dynein arms.

PubMed

Cho, Kyeong Jee; Noh, Shin Hye; Han, Soo Min; Choi, Won-Il; Kim, Hye-Youn; Yu, Seyoung; Lee, Joon Suk; Rim, John Hoon; Lee, Min Goo; Hildebrandt, Friedhelm; Gee, Heon Yung

2018-03-01

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.

Chlamydia trachomatis inclusion membrane protein CT850 interacts with the dynein light chain DYNLT1 (Tctex1).

PubMed

Mital, Jeffrey; Lutter, Erika I; Barger, Alexandra C; Dooley, Cheryl A; Hackstadt, Ted

2015-06-26

Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC. Published by Elsevier Inc.

A new mode of mitochondrial transport and polarized sorting regulated by Dynein, Milton and Miro.

PubMed

Melkov, Anna; Baskar, Raju; Alcalay, Yehonatan; Abdu, Uri

2016-11-15

Intrinsic cell microtubule (MT) polarity, together with molecular motors and adaptor proteins, determines mitochondrial polarized targeting and MT-dependent transport. In polarized cells, such as neurons, mitochondrial mobility and transport require the regulation of kinesin and dynein by two adaptor proteins, Milton and Miro. Recently, we found that dynein heavy chain 64C (Dhc64C) is the primary motor protein for both anterograde and retrograde transport of mitochondria in the Drosophila bristle. In this study, we show that a molecular lesion in the Dhc64C allele that reduced bristle mitochondrial velocity generated a variant that acts as a 'slow' dynein in an MT-gliding assay, indicating that dynein directly regulates mitochondrial transport. We also showed that in milton-RNAi flies, mitochondrial flux into the bristle shaft, but not velocity, was significantly reduced. Surprisingly, mitochondria retrograde flux, but not net velocity, was significantly decreased in miro-RNAi flies. We thus reveal a new mode of mitochondrial sorting in polarized cell growth, whereby bi-directional mitochondrial transport undertaken exclusively by dynein is regulated by Milton in the anterograde direction and by a Miro-dependent switch to the retrograde direction. © 2016. Published by The Company of Biologists Ltd.

A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

PubMed Central

Tang, Xiaohu; Seyb, Kathleen I.; Huang, Mickey; Schuman, Eli R.; Shi, Ping; Zhu, Haining; Glicksman, Marcie A.

2013-01-01

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells. PMID:22140121

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

PubMed Central

Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

2016-01-01

Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

Studying the protein organization of the postsynaptic density by a novel solid phase- and chemical cross-linking-based technology.

PubMed

Liu, Szu-Heng; Cheng, Huei-Hsuan; Huang, San-Yuan; Yiu, Pei-Chun; Chang, Yen-Chung

2006-06-01

Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules with their original abundances could provide information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslips. By using these beads, we determined the interior or superficial locations of five major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian central nervous system. The results indicate that alpha,beta-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, an hsp70 protein, alpha-internexin, dynamin, and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, kainate receptors, N-cadherin, beta-catenin, N-ethylmaleimide-sensitive factor, an hsc70 protein, and actin reside on the surface of the PSD. The results further suggest that the N-methyl-d-aspartate receptors and the alpha-subunits of calcium/calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD although with unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.

A split motor domain in a cytoplasmic dynein

PubMed Central

Straube, Anne; Enard, Wolfgang; Berner, Alexandra; Wedlich-Söldner, Roland; Kahmann, Regine; Steinberg, Gero

2001-01-01

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions. PMID:11566874

Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luthra, Priya; Jordan, David S.; Leung, Daisy W.

2015-03-04

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

Distinct roles of 1α and 1β heavy chains of the inner arm dynein I1 of Chlamydomonas flagella

PubMed Central

Toba, Shiori; Fox, Laura A.; Sakakibara, Hitoshi; Porter, Mary E.; Oiwa, Kazuhiro; Sale, Winfield S.

2011-01-01

The Chlamydomonas I1 dynein is a two-headed inner dynein arm important for the regulation of flagellar bending. Here we took advantage of mutant strains lacking either the 1α or 1β motor domain to distinguish the functional role of each motor domain. Single- particle electronic microscopic analysis confirmed that both the I1α and I1β complexes are single headed with similar ringlike, motor domain structures. Despite similarity in structure, however, the I1β complex has severalfold higher ATPase activity and microtubule gliding motility compared to the I1α complex. Moreover, in vivo measurement of microtubule sliding in axonemes revealed that the loss of the 1β motor results in a more severe impairment in motility and failure in regulation of microtubule sliding by the I1 dynein phosphoregulatory mechanism. The data indicate that each I1 motor domain is distinct in function: The I1β motor domain is an effective motor required for wild-type microtubule sliding, whereas the I1α motor domain may be responsible for local restraint of microtubule sliding. PMID:21148301

Selenium Deficiency Induces Autophagy in Immune Organs of Chickens.

PubMed

Khoso, Pervez Ahmed; Pan, Tingru; Wan, Na; Yang, Zijiang; Liu, Ci; Li, Shu

2017-05-01

The aim of the present study was to investigate the effects of selenium (Se) deficiency on autophagy-related genes and on ultrastructural changes in the spleen, bursa of Fabricius, and thymus of chickens. The Se deficiency group was fed a basal diet containing Se at 0.033 mg/kg and the control group was fed the same basal diet containing Se at 0.15 mg/kg. The messenger RNA (mRNA) levels of the autophagy genes microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin 1, dynein, autophagy associated gene 5 (ATG5), and target of rapamycin complex 1 (TORC1) were assessed using real-time qPCR. The protein levels of LC3-II, Beclin 1, and dynein were investigated using western blot analysis. Furthermore, the ultrastructure was observed using an electron microscope. The results indicated that spleen mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of LC3-II, Beclin 1, and dynein were increased in the Se deficiency group compared with the control group. In the bursa of Fabricius, the mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of Beclin 1 and dynein were increased; furthermore, the protein level of LC3-II was decreased in the Se deficiency group compared to the control group. In the thymus, the mRNA levels of LC3-I, Beclin 1, and ATG5 increased; the levels of LC3-II, dynein, and TORC1 were decreased; the protein level of Beclin 1 increased; and the levels of LC3-II and dynein decreased in the Se deficiency group compared to those in the control group. Further cellular morphological changes, such as autophagy vacuoles, autolysosomes, and lysosomal degradation, were observed in the spleen, bursa of Fabricius, and thymus of the Se-deficiency group. In summary, Se deficiency caused changes in autophagy-related genes, which increased the autophagic process and also caused structural damages to the immune organs of chickens.

Inhibition of microtubules and dynein rescues human immunodeficiency virus type 1 from owl monkey TRIMCyp-mediated restriction in a cellular context-specific fashion.

PubMed

Pawlica, Paulina; Dufour, Caroline; Berthoux, Lionel

2015-04-01

IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp. © 2015 The Authors.

Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription through Interaction with Its Integrase Protein

PubMed Central

Jayappa, Kallesh Danappa; Ao, Zhujun; Wang, Xiaoxia; Mouland, Andrew J.; Shekhar, Sudhanshu; Yang, Xi

2015-01-01

ABSTRACT In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs 52GQVD and 250VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral replication steps. PMID:25568209

S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner

PubMed Central

Grallert, Agnes; Beuter, Christoph; Craven, Rachel A.; Bagley, Steve; Wilks, Deepti; Fleig, Ursula; Hagan, Iain M.

2006-01-01

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.Δ and tip1.Δ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170. PMID:16951255

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport.

PubMed

Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R; Mans, Dorus A; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E C; Yap, Zhi Min; Letteboer, Stef J F; Taylor, S Paige; Herridge, Warren; Johnson, Colin A; Scambler, Peter J; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M; Beales, Philip L; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M; Witman, George B

2015-06-05

The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.

Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

PubMed

Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

2010-10-06

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caly, Leon; Kassouf, Vicki T.; Moseley, Gregory W.

Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72Lmore » mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. - Highlights: • HIV-1 Vpr utilizes the microtubule network to traffic towards the nucleus. • Mechanism relies on interaction between Vpr and dynein light chain protein DYNLT1. • Long-term non-progressor derived mutation (F72L) impairs this interaction. • Key residues in the vicinity of F72 contribute to interaction with DYNLT1.« less

Disruption in the autophagic process underlies the sensory neuropathy in dystonia musculorum mice.

PubMed

Ferrier, Andrew; De Repentigny, Yves; Lynch-Godrei, Anisha; Gibeault, Sabrina; Eid, Walaa; Kuo, Daniel; Zha, Xiaohui; Kothary, Rashmi

2015-01-01

A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dst(dt)) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dst(dt-Tg4) mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dst(dt-Tg4) sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dst(dt-Tg4) mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dst(dt-Tg4) mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dst(dt-Tg4) dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dst(dt) pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis.

Disruption in the autophagic process underlies the sensory neuropathy in dystonia musculorum mice

PubMed Central

Ferrier, Andrew; De Repentigny, Yves; Lynch-Godrei, Anisha; Gibeault, Sabrina; Eid, Walaa; Kuo, Daniel; Zha, Xiaohui; Kothary, Rashmi

2015-01-01

A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dstdt) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dstdt-Tg4 mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dstdt-Tg4 sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dstdt-Tg4 mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dstdt-Tg4 mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dstdt-Tg4 dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dstdt pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis. PMID:26043942

Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

PubMed Central

Martin, R.; Walther, A.; Wendland, J.

2004-01-01

Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1. PMID:15590831

Structural atlas of dynein motors at atomic resolution.

PubMed

Toda, Akiyuki; Tanaka, Hideaki; Kurisu, Genji

2018-04-01

Dynein motors are biologically important bio-nanomachines, and many atomic resolution structures of cytoplasmic dynein components from different organisms have been analyzed by X-ray crystallography, cryo-EM, and NMR spectroscopy. This review provides a historical perspective of structural studies of cytoplasmic and axonemal dynein including accessory proteins. We describe representative structural studies of every component of dynein and summarize them as a structural atlas that classifies the cytoplasmic and axonemal dyneins. Based on our review of all dynein structures in the Protein Data Bank, we raise two important points for understanding the two types of dynein motor and discuss the potential prospects of future structural studies.

Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity

PubMed Central

Kato, Yuiko; Ishiguro-Oonuma, Toshina; Udagawa, Chihiro; Rungsuriyawiboon, Oumaporn; Azakami, Daigo; Michishita, Masaki; Ariyoshi, Yuichi; Ueki, Hideo; Nasu, Yasutomo; Kumon, Hiromi; Watanabe, Masami; Omi, Toshinori

2016-01-01

REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling. PMID:26658102

Dynactin functions as both a dynamic tether and brake during dynein-driven motility

NASA Astrophysics Data System (ADS)

Ayloo, Swathi; Lazarus, Jacob E.; Dodda, Aditya; Tokito, Mariko; Ostap, E. Michael; Holzbaur, Erika L. F.

2014-09-01

Dynactin is an essential cofactor for most cellular functions of the microtubule motor cytoplasmic dynein, but the mechanism by which dynactin activates dynein remains unclear. Here we use single molecule approaches to investigate dynein regulation by the dynactin subunit p150Glued. We investigate the formation and motility of a dynein-p150Glued co-complex using dual-colour total internal reflection fluorescence microscopy. p150Glued recruits and tethers dynein to the microtubule in a concentration-dependent manner. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of p150Glued decreases the detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

NudEL targets dynein to microtubule ends through LIS1

PubMed Central

Li, Jun; Lee, Wei-Lih; Cooper, John A.

2006-01-01

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin1, LIS1 (refs 2, 3) and NudEL (NudE-like)2,4–8. Here, we identify a NUDEL homologue in budding yeast and name it Ndl1. The ndl1Δ null mutant shows decreased targeting of dynein to microtubule plus ends, an essential element of the model for dynein function. We find that Ndl1 regulates dynein targeting through LIS1, with which it interacts biochemically, but not through CLIP170, another plus-end protein involved in dynein targeting9. Ndl1 is found at far fewer microtubule ends than are LIS1 and dynein. However, when Ndl1 is present at a plus end, the molar amount of Ndl1 approaches that of LIS1 and dynein. We propose a model in which Ndl1 binds transiently to the plus end to promote targeting of LIS1, which cooperatively recruits dynein. PMID:15965467

NudEL targets dynein to microtubule ends through LIS1.

PubMed

Li, Jun; Lee, Wei-Lih; Cooper, John A

2005-07-01

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin, LIS1 (refs 2, 3) and NudEL (NudE-like). Here, we identify a NUDEL homologue in budding yeast and name it Ndl1. The ndl1delta null mutant shows decreased targeting of dynein to microtubule plus ends, an essential element of the model for dynein function. We find that Ndl1 regulates dynein targeting through LIS1, with which it interacts biochemically, but not through CLIP170, another plus-end protein involved in dynein targeting. Ndl1 is found at far fewer microtubule ends than are LIS1 and dynein. However, when Ndl1 is present at a plus end, the molar amount of Ndl1 approaches that of LIS1 and dynein. We propose a model in which Ndl1 binds transiently to the plus end to promote targeting of LIS1, which cooperatively recruits dynein.

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

PubMed Central

Lammers, Lindsay G.

2015-01-01

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal. PMID:26483554

Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein

PubMed Central

2012-01-01

Background Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150Glued), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11). Results To reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons. Conclusions The dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events. PMID:22726940

Dync1h1 Mutation Causes Proprioceptive Sensory Neuron Loss and Impaired Retrograde Axonal Transport of Dorsal Root Ganglion Neurons.

PubMed

Zhao, Jing; Wang, Yi; Xu, Huan; Fu, Yuan; Qian, Ting; Bo, Deng; Lu, Yan-Xin; Xiong, Yi; Wan, Jun; Zhang, Xiang; Dong, Qiang; Chen, Xiang-Jun

2016-07-01

Sprawling (Swl) is a radiation-induced mutation which has been identified to have a nine base pair deletion in dynein heavy chain 1 (DYNC1H1: encoded by a single gene Dync1h1). This study is to investigate the phenotype and the underlying mechanism of the Dync1h1 mutant. To display the phenotype of Swl mutant mice, we examined the embryos of homozygous (Swl/Swl) and heterozygous (Swl/+) mice and their postnatal dorsal root ganglion (DRG) of surviving Swl/+ mice. The Swl/+ mice could survive for a normal life span, while Swl/Swl could only survive till embryonic (E) 8.5 days. Excessive apoptosis of Swl/+ DRG neurons was revealed during E11.5-E15.5 days, and the peak rate was at E13.5 days. In vitro study of mutated DRG neurons showed impaired retrograde transport of dynein-driven nerve growth factor (NGF). Mitochondria, another dynein-driven cargo, demonstrated much slower retrograde transport velocity in Swl/+ neurons than in wild-type (WT) neurons. Nevertheless, the Swl, Loa, and Cra mutations did not affect homodimerization of DYNC1H1. The Swl/Swl mutation of Dync1h1 gene led to embryonic mal-development and lethality, whereas the Swl/+ DRG neurons demonstrated deficient retrograde transport in dynein-driven cargos and excessive apoptosis during mid- to late-developmental stages. The underlying mechanism of the mutation may not be due to impaired homodimerization of DYNC1H1. © 2016 John Wiley & Sons Ltd.

Ciliobrevins as tools for studying dynein motor function

PubMed Central

Roossien, Douglas H.; Miller, Kyle E.; Gallo, Gianluca

2015-01-01

Dyneins are a small class of molecular motors that bind to microtubules and walk toward their minus ends. They are essential for the transport and distribution of organelles, signaling complexes and cytoskeletal elements. In addition dyneins generate forces on microtubule arrays that power the beating of cilia and flagella, cell division, migration and growth cone motility. Classical approaches to the study of dynein function in axons involve the depletion of dynein, expression of mutant/truncated forms of the motor, or interference with accessory subunits. By necessity, these approaches require prolonged time periods for the expression or manipulation of cellular dynein levels. With the discovery of the ciliobrevins, a class of cell permeable small molecule inhibitors of dynein, it is now possible to acutely disrupt dynein both globally and locally. In this review, we briefly summarize recent work using ciliobrevins to inhibit dynein and discuss the insights ciliobrevins have provided about dynein function in various cell types with a focus on neurons. We temper this with a discussion of the need for studies that will elucidate the mechanism of action of ciliobrevin and as well as the need for experiments to further analyze the specificity of ciliobreviens for dynein. Although much remains to be learned about ciliobrevins, these small molecules are proving themselves to be valuable novel tools to assess the cellular functions of dynein. PMID:26217180

Lissencephaly-1 is a context-dependent regulator of the human dynein complex

PubMed Central

Baumbach, Janina; Murthy, Andal; McClintock, Mark A; Dix, Carly I; Zalyte, Ruta; Hoang, Ha Thi; Bullock, Simon L

2017-01-01

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo. DOI: http://dx.doi.org/10.7554/eLife.21768.001 PMID:28406398

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms*

PubMed Central

Radnai, László; Rapali, Péter; Hódi, Zsuzsa; Süveges, Dániel; Molnár, Tamás; Kiss, Bence; Bécsi, Bálint; Erdödi, Ferenc; Buday, László; Kardos, József; Kovács, Mihály; Nyitray, László

2010-01-01

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with Kd values of 9 and 40 μm, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: Kd values of 37 and 3.5 nm for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent Kd value (3 μm). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network. PMID:20889982

Tight coupling between nucleus and cell migration through the perinuclear actin cap

PubMed Central

Kim, Dong-Hwee; Cho, Sangkyun; Wirtz, Denis

2014-01-01

ABSTRACT Although eukaryotic cells are known to alternate between ‘advancing’ episodes of fast and persistent movement and ‘hesitation’ episodes of low speed and low persistence, the molecular mechanism that controls the dynamic changes in morphology, speed and persistence of eukaryotic migratory cells remains unclear. Here, we show that the movement of the interphase nucleus during random cell migration switches intermittently between two distinct modes – rotation and translocation – that follow with high fidelity the sequential rounded and elongated morphologies of the nucleus and cell body, respectively. Nuclear rotation and translocation mediate the stop-and-go motion of the cell through the dynamic formation and dissolution, respectively, of the contractile perinuclear actin cap, which is dynamically coupled to the nuclear lamina and the nuclear envelope through LINC complexes. A persistent cell movement and nuclear translocation driven by the actin cap are halted following the disruption of the actin cap, which in turn allows the cell to repolarize for its next persistent move owing to nuclear rotation mediated by cytoplasmic dynein light intermediate chain 2. PMID:24639463

The interaction of flavivirus M protein with light chain Tctex-1 of human dynein plays a role in late stages of virus replication.

PubMed

Brault, Jean-Baptiste; Kudelko, Mateusz; Vidalain, Pierre-Olivier; Tangy, Frédéric; Desprès, Philippe; Pardigon, Nathalie

2011-09-01

The role of the membrane protein (prM/M) in flavivirus life cycle remains unclear. Here, we identified a cellular interactor to the 40-residue-long ectodomain of prM/M (ectoM) using a yeast two-hybrid screen against a human cDNA library and GST pull-down assays. We showed that dynein light chain Tctex-1 interacts with the ectoM of dengue 1-4, West Nile, and Japanese encephalitis flaviviruses. No interaction was found with yellow fever and tick-borne flaviviruses. This interaction is highly specific since a single amino-acid change in the ectoM abrogates the interaction with Tctex-1. To understand the role of this interaction, silencing of Tctex-1 using siRNA was performed prior to infection. A significant decrease in progeny production was observed for dengue and West Nile viruses. Silencing Tctex-1 inhibited the production of recombinant dengue subviral particles (RSPs). Thus Tctex-1 may play a role in late stages of viral replication through its interaction with the membrane protein. Copyright © 2011 Elsevier Inc. All rights reserved.

BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

PubMed Central

Splinter, Daniël; Razafsky, David S.; Schlager, Max A.; Serra-Marques, Andrea; Grigoriev, Ilya; Demmers, Jeroen; Keijzer, Nanda; Jiang, Kai; Poser, Ina; Hyman, Anthony A.; Hoogenraad, Casper C.; King, Stephen J.; Akhmanova, Anna

2012-01-01

Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors. PMID:22956769

Bidirectional helical motility of cytoplasmic dynein around microtubules

PubMed Central

Can, Sinan; Dewitt, Mark A; Yildiz, Ahmet

2014-01-01

Cytoplasmic dynein is a molecular motor responsible for minus-end-directed cargo transport along microtubules (MTs). Dynein motility has previously been studied on surface-immobilized MTs in vitro, which constrains the motors to move in two dimensions. In this study, we explored dynein motility in three dimensions using an MT bridge assay. We found that dynein moves in a helical trajectory around the MT, demonstrating that it generates torque during cargo transport. Unlike other cytoskeletal motors that produce torque in a specific direction, dynein generates torque in either direction, resulting in bidirectional helical motility. Dynein has a net preference to move along a right-handed helical path, suggesting that the heads tend to bind to the closest tubulin binding site in the forward direction when taking sideways steps. This bidirectional helical motility may allow dynein to avoid roadblocks in dense cytoplasmic environments during cargo transport. DOI: http://dx.doi.org/10.7554/eLife.03205.001 PMID:25069614

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

PubMed

Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C

2015-11-26

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Bim Inhibits Autophagy by Recruiting Beclin 1 to Microtubules

PubMed Central

Luo, Shouqing; Garcia-Arencibia, Moises; Zhao, Rui; Puri, Claudia; Toh, Pearl P.C.; Sadiq, Oana; Rubinsztein, David C.

2012-01-01

Summary Bim is a proapoptotic BH3-only Bcl-2 family member. In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites. PMID:22742832

The human cytoplasmic dynein interactome reveals novel activators of motility

PubMed Central

Redwine, William B; DeSantis, Morgan E; Hollyer, Ian; Htet, Zaw Min; Tran, Phuoc Tien; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Reck-Peterson, Samara L

2017-01-01

In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome by attaching a promiscuous biotin ligase (‘BioID’) to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic dynein/dynactin complex in living cells. To achieve maximal motile activity and to bind its cargos, human dynein/dynactin requires ‘activators’, of which only five have been described. We developed methods to identify new activators in our BioID data, and discovered that ninein and ninein-like are a new family of dynein activators. Analysis of the protein interactomes for six activators, including ninein and ninein-like, suggests that each dynein activator has multiple cargos. DOI: http://dx.doi.org/10.7554/eLife.28257.001 PMID:28718761

Lis1 regulates dynein by sterically blocking its mechanochemical cycle

PubMed Central

Toropova, Katerina; Zou, Sirui; Roberts, Anthony J; Redwine, William B; Goodman, Brian S; Reck-Peterson, Samara L; Leschziner, Andres E

2014-01-01

Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein–Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the ‘linker’, from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle. DOI: http://dx.doi.org/10.7554/eLife.03372.001 PMID:25380312

Reconstitution of dynein transport to the microtubule plus end by kinesin

PubMed Central

Roberts, Anthony J; Goodman, Brian S; Reck-Peterson, Samara L

2014-01-01

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track. DOI: http://dx.doi.org/10.7554/eLife.02641.001 PMID:24916158

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

PubMed Central

Tan, Kaeling; Roberts, Anthony J.; Chonofsky, Mark; Egan, Martin J.; Reck-Peterson, Samara L.

2014-01-01

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance. PMID:24403603

Dynein-mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

PubMed Central

Fink, Gero; Schuchardt, Isabel; Colombelli, Julien; Stelzer, Ernst; Steinberg, Gero

2006-01-01

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches ∼2 μm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex. PMID:17024185

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

PubMed Central

Caviston, Juliane P.; Zajac, Allison L.; Tokito, Mariko; Holzbaur, Erika L.F.

2011-01-01

Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles. PMID:21169558

Effect of fuel concentration and force on collective transport by a team of dynein motors

PubMed Central

Takshak, Anjneya; Roy, Tanushree; Tandaiya, Parag

2016-01-01

Abstract Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces. PMID:27727483

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes

PubMed Central

Bielska, Ewa; Schuster, Martin; Roger, Yvonne; Berepiki, Adokiye; Soanes, Darren M.; Talbot, Nicholas J.

2014-01-01

Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3– and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein. PMID:24637326

Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin DependentV⃞

PubMed Central

Zhang, Jun; Li, Shihe; Fischer, Reinhard; Xiang, Xin

2003-01-01

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF. PMID:12686603

Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment▿

PubMed Central

Ramanathan, Harish N.; Chung, Dong-Hoon; Plane, Steven J.; Sztul, Elizabeth; Chu, Yong-kyu; Guttieri, Mary C.; McDowell, Michael; Ali, Georgia; Jonsson, Colleen B.

2007-01-01

In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region. PMID:17537852

Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation

PubMed Central

Markus, Steven M.; Lee, Wei-Lih

2011-01-01

During animal development, microtubules (MTs) play a major role in directing cellular and subcellular patterning, impacting cell polarization and subcellular organization, thereby affecting cell fate determination and tissue architecture. In particular, when progenitor cells divide asymmetrically along an anterior-posterior or apical-basal axis, MTs must coordinate the position of the mitotic spindle with the site of cell division to ensure normal distribution of cell fate determinants and equal sequestration of genetic material into the two daughter cells. Emerging data from diverse model systems have led to the prevailing view that, during mitotic spindle positioning, polarity cues at the cell cortex signal for the recruitment of NuMA and the minus-end directed MT motor cytoplasmic dynein.1 The NuMA/dynein complex is believed to connect, in turn, to the mitotic spindle via astral MTs, thus aligning and tethering the spindle, but how this connection is achieved faithfully is unclear. Do astral MTs need to search for and then capture cortical NuMA/dynein? How does dynein capture the astral MTs emanating from the correct spindle pole? Recently, using the classical model of asymmetric cell division—budding yeast S. cerevisiae—we successfully demonstrated that astral MTs assume an active role in cortical dynein targeting, in that astral MTs utilize their distal plus ends to deliver dynein to the daughter cell cortex, the site where dynein activity is needed to perform its spindle alignment function. This observation introduced the novel idea that, during mitotic spindle orientation processes, polarity cues at the cell cortex may actually signal to prime the cortical receptors for MT-dependent dynein delivery. This model is consistent with the observation that dynein/dynactin accumulate prominently at the astral MT plus ends during metaphase in a wide range of cultured mammalian cells. PMID:22754610

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events.

PubMed

Motil, Jennifer; Chan, Walter K-H; Dubey, Maya; Chaudhury, Pulkit; Pimenta, Aurea; Chylinski, Teresa M; Ortiz, Daniela T; Shea, Thomas B

2006-05-01

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin. Copyright 2006 Wiley-Liss, Inc.

Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

NASA Astrophysics Data System (ADS)

Namdeo, S.; Onck, P. R.

2016-10-01

Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins.

PubMed

Namdeo, S; Onck, P R

2016-10-01

Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest–specific gene product

PubMed Central

Rupp, Gerald; Porter, Mary E.

2003-01-01

The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest–specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures. PMID:12847082

Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms

PubMed Central

Yamamoto, Ryosuke; Alford, Lea M.; Ide, Takahiro; Owa, Mikito; Hwang, Juyeon; Inaba, Kazuo; James, Noliyanda; Ishikawa, Takashi

2017-01-01

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly. PMID:28892495

Dynein's Network of Chemomechanical Motor Cycles

NASA Astrophysics Data System (ADS)

Shen, Weibo; Wang, Ziqing; Wang, Guodong

2012-07-01

An eight-state network model of dynein's chemomechanical motor cycles is developed, in which the states of an effective single dynein head are represented by the number of ATP binding at the primary site and the number of ATP binding at other three secondary sites. The binding and unbinding of ATP, as well as the hydrolysis of ATP and the reverse process, are characterized by transition rates between certain states. Our results show that the stall force of dynein increases fast with ATP up to 1 mM ATP, beyond which it increases slowly to a saturated value, and that load and ATP concentration can adjust the step size of dynein, i.e., dynein can shift gears according to conditions. These results are in agreement with experiments [R. Mallik, B. C. Carter, S. A. Lex, S. J. King and S. P. Gross, Nature 427, 649 (2004)].

The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids.

PubMed

Osseman, Quentin; Gallucci, Lara; Au, Shelly; Cazenave, Christian; Berdance, Elodie; Blondot, Marie-Lise; Cassany, Aurélia; Bégu, Dominique; Ragues, Jessica; Aknin, Cindy; Sominskaya, Irina; Dishlers, Andris; Rabe, Birgit; Anderson, Fenja; Panté, Nelly; Kann, Michael

2018-03-01

Hepatitis B virus (HBV) has a DNA genome but replicates within the nucleus by reverse transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are a major target for T cell-mediated immune responses. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal. We used virion-derived capsids containing mature rcDNA (matC) and empty capsids (empC). RNA-containing capsids (rnaC) were used as a control. The investigations comprised pull-down assays for identification of cellular interaction partners, immune fluorescence microscopy for their colocalization and electron microscopy after microinjection to determine their biological significance. matC and empC underwent active transport through the cytoplasm towards the nucleus, while rnaC was poorly transported. We identified the dynein light chain LL1 as a functional interaction partner linking capsids to the dynein motor complex and showed that there is no compensatory transport pathway. Using capsid and dynein LL1 mutants we characterized the required domains on the capsid and LL1. This is the first investigation on the detailed molecular mechanism of how matC pass the cytoplasm upon infection and how empC can be actively removed from the cytoplasm into the nucleus. Considering that hepatocytes with cytoplasmic capsids are better recognized by the T cells, we hypothesize that targeting capsid DynLL1-interaction will not only block HBV infection but also stimulate elimination of infected cells. In this study, we identified the molecular details of HBV translocation through the cytoplasm. Our evidence offers a new drug target which could not only inhibit infection but also stimulate immune clearance of HBV infected cells. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

A novel mouse model carrying a human cytoplasmic dynein mutation shows motor behavior deficits consistent with Charcot-Marie-Tooth type 2O disease.

PubMed

Sabblah, Thywill T; Nandini, Swaran; Ledray, Aaron P; Pasos, Julio; Calderon, Jami L Conley; Love, Rachal; King, Linda E; King, Stephen J

2018-01-29

Charcot-Marie-Tooth disease (CMT) is a peripheral neuromuscular disorder in which axonal degeneration causes progressive loss of motor and sensory nerve function. The loss of motor nerve function leads to distal muscle weakness and atrophy, resulting in gait problems and difficulties with walking, running, and balance. A mutation in the cytoplasmic dynein heavy chain (DHC) gene was discovered to cause an autosomal dominant form of the disease designated Charcot-Marie-Tooth type 2 O disease (CMT2O) in 2011. The mutation is a single amino acid change of histidine into arginine at amino acid 306 (H306R) in DHC. In order to understand the onset and progression of CMT2, we generated a knock-in mouse carrying the corresponding CMT2O mutation (H304R/+). We examined H304R/+ mouse cohorts in a 12-month longitudinal study of grip strength, tail suspension, and rotarod assays. H304R/+ mice displayed distal muscle weakness and loss of motor coordination phenotypes consistent with those of individuals with CMT2. Analysis of the gastrocnemius of H304R/+ male mice showed prominent defects in neuromuscular junction (NMJ) morphology including reduced size, branching, and complexity. Based on these results, the H304R/+ mouse will be an important model for uncovering functions of dynein in complex organisms, especially related to CMT onset and progression.

Monte Carlo modeling of single-molecule cytoplasmic dynein.

PubMed

Singh, Manoranjan P; Mallik, Roop; Gross, Steven P; Yu, Clare C

2005-08-23

Molecular motors are responsible for active transport and organization in the cell, underlying an enormous number of crucial biological processes. Dynein is more complicated in its structure and function than other motors. Recent experiments have found that, unlike other motors, dynein can take different size steps along microtubules depending on load and ATP concentration. We use Monte Carlo simulations to model the molecular motor function of cytoplasmic dynein at the single-molecule level. The theory relates dynein's enzymatic properties to its mechanical force production. Our simulations reproduce the main features of recent single-molecule experiments that found a discrete distribution of dynein step sizes, depending on load and ATP concentration. The model reproduces the large steps found experimentally under high ATP and no load by assuming that the ATP binding affinities at the secondary sites decrease as the number of ATP bound to these sites increases. Additionally, to capture the essential features of the step-size distribution at very low ATP concentration and no load, the ATP hydrolysis of the primary site must be dramatically reduced when none of the secondary sites have ATP bound to them. We make testable predictions that should guide future experiments related to dynein function.

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells.

PubMed

Baffet, Alexandre D; Hu, Daniel J; Vallee, Richard B

2015-06-22

Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2 via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell-cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell-cycle regulated and identify the trigger mechanism for apical nuclear migration in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

NASA Astrophysics Data System (ADS)

Feofilova, Maria; Howard, Jonathon

Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

Chemical and thermal modulation of molecular motor activities

NASA Astrophysics Data System (ADS)

Hong, Weili

Molecular motors of kinesin and dynein families are responsible for various intracellular activities, from long distance movement of organelles, vesicles, protein complexes, and mRNAs to powering mitotic processes. They can take nanometer steps using chemical energy from the hydrolysis of ATP (adenosine triphosphate), and their dysfunction is involved in many neurodegenerative diseases that require long distance transport of cargos. Here I report on the study of the properties of molecular motors at a single-molecule level using optical trappings. I first studied the inhibition properties of kinesin motors by marine natural compound adociasulfates. I showed that adociasulfates compete with microtubules for binding to kinesins and thus inhibit kinesins' activity. Although adociasulfates are a strong inhibitor for all kinesin members, they show a much higher inhibition effect for conventional kinesins than for mitotic kinesins. Thus adociasulfates can be used to specifically inhibit conventional kinesins. By comparing the inhibition of kinesins by two structurally similar adociasulfates, one can see that the negatively charged sulfate residue of adociasulfates can be replaced by other negative residues and thus make it possible for adociasulfate-derived compounds to be more cell permeable. Kinesins and dyneins move cargos towards opposite directions along a microtubule. Cargos with both kinesins and dyneins attached often move bidirectionally due to undergoing a tug-of-war between the oppositely moving kinesin and dynein motors. Here I studied the effect of temperature on microtubule-based kinesin and dynein motor transport. While kinesins' and dyneins' velocities are closely matched above 15 °C, below this temperature the dyneins' velocity decreases much faster than the kinesins'. The kinesins' and dyneins' forces do not measurably change with temperature. The results suggest that temperature has significant effects on bidirectional transport and can be used to probe tug-of-war mechanism between kinesins and dyneins.

Lis1 acts as a "clutch" between the ATPase and microtubule-binding domains of the dynein motor.

PubMed

Huang, Julie; Roberts, Anthony J; Leschziner, Andres E; Reck-Peterson, Samara L

2012-08-31

The lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dynein's AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a "clutch" that prevents dynein's ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments. Copyright © 2012 Elsevier Inc. All rights reserved.

Lis1 Acts as a “Clutch” between the ATPase and Microtubule-Binding Domains of the Dynein Motor

PubMed Central

Huang, Julie; Roberts, Anthony J.; Leschziner, Andres E.; Reck-Peterson, Samara L.

2012-01-01

Summary The lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dynein's AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a “clutch” that prevents dynein's ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments. PMID:22939623

Dynein-deficient flagella respond to increased viscosity with contrasting changes in power and recovery strokes.

PubMed

Wilson, Kate S; Gonzalez, Olivia; Dutcher, Susan K; Bayly, Philip V

2015-09-01

Changes in the flagellar waveform in response to increased viscosity were investigated in uniflagellate mutants of Chlamydomonas reinhardtii. We hypothesized that the waveforms of mutants lacking different dynein arms would change in different ways as viscosity was increased, and that these variations would illuminate the feedback pathways from force to dynein activity. Previous studies have investigated the effects of viscosity on cell body motion, propulsive force, and power in different mutants, but the effect on waveform has not yet been fully characterized. Beat frequency decreases with viscosity in wild-type uniflagellate (uni1) cells, and outer dynein arm deficient (oda2) mutants. In contrast, the inner dynein arm mutant ida1 (lacking I1/f) maintains beat frequency at high viscosity but alters its flagellar waveform more than either wild-type or oda2. The ida1 waveform is narrower than wild-type, primarily due to an abbreviated recovery stroke; this difference is amplified at high viscosity. The oda2 mutant in contrast, maintains a consistent waveform at high and low viscosity with a slightly longer power stroke than wild-type. Analysis of the delays and shear displacements between bends suggest that direct force feedback in the outer dynein arm system may initiate switching of dynein activity. In contrast, I1/f dynein appears to delay switching, most markedly at the initiation of the power stroke, possibly by controlling inter-doublet separation. © 2015 Wiley Periodicals, Inc.

Dynein-deficient flagella respond to increased viscosity with contrasting changes in power and recovery strokes

PubMed Central

Wilson, Kate S.; Gonzalez, Olivia; Dutcher, Susan K.; Bayly, P.V.

2015-01-01

Changes in the flagellar waveform in response to increased viscosity were investigated in uniflagellate mutants of Chlamydomonas reinhardtii. We hypothesized that the waveforms of mutants lacking different dynein arms would change in different ways as viscosity was increased, and that these variations would illuminate the feedback pathways from force to dynein activity. Previous studies have investigated the effects of viscosity on cell body motion, propulsive force, and power in different mutants, but the effect on waveform has not yet been fully characterized. Beat frequency decreases with viscosity in wild-type uniflagellate (uni1) cells, and outer dynein arm deficient (oda2) mutants. In contrast, the inner dynein arm mutant ida1 (lacking I1/f) maintains beat frequency at high viscosity but alters its flagellar waveform more than either wild-type or oda2. The ida1 waveform is narrower than wild-type, primarily due to an abbreviated recovery stroke; this difference is amplified at high viscosity. The oda2 mutant in contrast, maintains a consistent waveform at high and low viscosity with a slightly longer power stroke than wild-type. Analysis of the delays and shear displacements between bends suggest that direct force feedback in the outer dynein arm system may initiate switching of dynein activity. In contrast, I1/f dynein appears to delay switching, most markedly at the initiation of the power stroke, possibly by controlling inter-doublet separation. PMID:26314933

Lipid Rafts Assemble Dynein Ensembles.

PubMed

Nirschl, Jeffrey J; Ghiretti, Amy E; Holzbaur, Erika L F

2016-05-01

New work by Rai et al. identifies a novel mechanism regulating phagosome transport in cells: the clustering of dynein motors into lipid microdomains, leading to enhanced unidirectional motility. Clustering may be especially important for dynein, a motor that works most efficiently in teams. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stopped in its tracks: negative regulation of the dynein motor by the yeast protein She1

PubMed Central

Moore, Jeffrey K.

2013-01-01

Summary How do cells direct the microtubule motor protein dynein to move cellular components to the right place at the right time? Recent studies in budding yeast shed light on a new mechanism for directing dynein, involving the protein She1. She1 restricts where and when dynein moves the nucleus and mitotic spindle. Experiments with purified proteins show that She1 binds to microtubules and inhibits dynein by stalling the motor on its track. Here I describe what we have learned so far about She1, based on a combination of genetic, cell biology, and biophysical approaches. These findings set the stage for further interrogation of the She1 mechanism, and raise the question of whether similar mechanisms exist in other species. PMID:23666903

Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the Spindle Checkpoint

PubMed Central

Yang, Zhenye; Tulu, U. Serdar; Wadsworth, Patricia; Rieder, Conly L.

2008-01-01

Summary During mitosis, the motor molecule cytoplasmic dynein plays key direct and indirect roles in organizing microtubules (MTs) into a functional spindle. At this time, dynein is also recruited to kinetochores, but its role or roles at these organelles remain vague, partly because inhibiting dynein globally disrupts spindle assembly [1-4]. However, dynein can be selectively depleted from kinetochores by disruption of ZW10 [5], and recent studies with this approach conclude that kinetochore-associated dynein (KD) functions to silence the spindle-assembly checkpoint (SAC) [6]. Here we use dynein-antibody microinjection and the RNAi of ZW10 to explore the role of KD in chromosome behavior during mitosis in mammals. We find that depleting or inhibiting KD prevents the rapid poleward motion of attaching kinetochores but not kinetochore fiber (K fiber) formation. However, after kinetochores attach to the spindle, KD is required for stabilizing kinetochore MTs, which it probably does by generating tension on the kinetochore, and in its absence, chromosome congression is defective. Finally, depleting KD reduces the velocity of anaphase chromosome motion by ∼40%, without affecting the rate of poleward MT flux. Thus, in addition to its role in silencing the SAC, KD is important for forming and stabilizing K fibers and in powering chromosome motion. PMID:17509882

Cytoplasmic dynein binding, run length, and velocity are guided by long-range electrostatic interactions

PubMed Central

Li, Lin; Alper, Joshua; Alexov, Emil

2016-01-01

Dyneins are important molecular motors involved in many essential biological processes, including cargo transport along microtubules, mitosis, and in cilia. Dynein motility involves the coupling of microtubule binding and unbinding to a change in the configuration of the linker domain induced by ATP hydrolysis, which occur some 25 nm apart. This leaves the accuracy of dynein stepping relatively inaccurate and susceptible to thermal noise. Using multi-scale modeling with a computational focusing technique, we demonstrate that the microtubule forms an electrostatic funnel that guides the dynein’s microtubule binding domain (MTBD) as it finally docks to the precise, keyed binding location on the microtubule. Furthermore, we demonstrate that electrostatic component of the MTBD’s binding free energy is linearly correlated with the velocity and run length of dynein, and we use this linearity to predict the effect of mutating each glutamic and aspartic acid located in MTBD domain to alanine. Lastly, we show that the binding of dynein to the microtubule is associated with conformational changes involving several helices, and we localize flexible hinge points within the stalk helices. Taken all together, we demonstrate that long range electrostatic interactions bring a level of precision to an otherwise noisy dynein stepping process. PMID:27531742

Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration

PubMed Central

DeZwaan, Todd M.; Ellingson, Eric; Pellman, David; Roof, David M.

1997-01-01

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus. PMID:9281581

Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores

PubMed Central

Kasuboski, James M.; Bader, Jason R.; Vaughan, Patricia S.; Tauhata, Sinji B. F.; Winding, Michael; Morrissey, Meghan A.; Joyce, Michelle V.; Boggess, William; Vos, Larissa; Chan, Gordon K.; Hinchcliffe, Edward H.; Vaughan, Kevin T.

2011-01-01

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase. PMID:21775627

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

PubMed Central

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O.; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-01-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim–Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. PMID:28982759

Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo.

PubMed

Takazaki, Hiroko; Liu, Zhongmei; Jin, Mingyue; Kamiya, Ritsu; Yasunaga, Takuo

2010-07-01

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, alpha-, beta-, and gamma-HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross-sectional electron micrographs of wild-type and single HC-disruption mutants were compared and statistically analyzed. While the alpha-HC mutant displayed an OAD of lower density, which was attributed to a lack of alpha-HC, the OAD of beta- and gamma-HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of alpha-HC. The lack of beta-HC induced mislocalization of alpha-HC, while a disruption of the gamma-HC gene resulted in the synchronized movement of alpha-HC and beta-HC in the manners for stacking. Interestingly, using cryo-electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to gamma-HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo-electron tomography and a bouquet structure on mica in deep-etch replica images. These results suggest that gamma-HC supports the tight stacking arrangement of inter or intra alpha-/beta-HC to facilitate the proper functioning of OAD. 2010 Wiley-Liss, Inc.

Tubulin Glutamylation Regulates Ciliary Motility by Altering Inner Dynein Arm Activity

PubMed Central

Suryavanshi, Swati; Eddé, Bernard; Fox, Laura A.; Guerrero, Stella; Hard, Robert; Hennessey, Todd; Kabi, Amrita; Malison, David; Pennock, David; Sale, Winfield S.; Wloga, Dorota; Gaertig, Jacek

2010-01-01

SUMMMARY How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by post-translational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this PTM, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms. PMID:20189389

Evidence for the Involvement of Lfc and Tctex-1 in Axon Formation

PubMed Central

Conde, Cecilia; Arias, Cristina; Robin, Maria; Li, Aiqun; Saito, Masaki; Chuang, Jen-Zen; Nairn, Angus C.; Sung, Ching-Hwa; Cáceres, Alfredo

2013-01-01

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation. PMID:20463241

Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action

PubMed Central

Steinman, Jonathan B; Santarossa, Cristina C; Miller, Rand M; Yu, Lola S; Serpinskaya, Anna S; Furukawa, Hideki; Morimoto, Sachie; Tanaka, Yuta; Nishitani, Mitsuyoshi; Asano, Moriteru; Zalyte, Ruta; Ondrus, Alison E; Johnson, Alex G; Ye, Fan; Nachury, Maxence V; Fukase, Yoshiyuki; Aso, Kazuyoshi; Foley, Michael A; Gelfand, Vladimir I; Chen, James K; Carter, Andrew P; Kapoor, Tarun M

2017-01-01

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition. DOI: http://dx.doi.org/10.7554/eLife.25174.001 PMID:28524820

CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

PubMed Central

Panizzi, Jennifer R.; Becker-Heck, Anita; Castleman, Victoria H.; Al-Mutairi, Dalal; Liu, Yan; Loges, Niki T.; Pathak, Narendra; Austin-Tse, Christina; Sheridan, Eamonn; Schmidts, Miriam; Olbrich, Heike; Werner, Claudius; Häffner, Karsten; Hellman, Nathan; Chodhari, Rahul; Gupta, Amar; Kramer-Zucker, Albrecht; Olale, Felix; Burdine, Rebecca D.; Schier, Alexander F.; O’Callaghan, Christopher; Chung, Eddie MK; Reinhardt, Richard; Mitchison, Hannah M.; King, Stephen M.; Omran, Heymut; Drummond, Iain A.

2012-01-01

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality1. Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhanstn222 (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia. PMID:22581229

Axonal autophagosomes recruit dynein for retrograde transport through fusion with late endosomes

PubMed Central

Cheng, Xiu-Tang; Zhou, Bing; Lin, Mei-Yao; Cai, Qian

2015-01-01

Efficient degradation of autophagic vacuoles (AVs) via lysosomes is an important cellular homeostatic process. This is particularly challenging for neurons because mature acidic lysosomes are relatively enriched in the soma. Although dynein-driven retrograde transport of AVs was suggested, a fundamental question remains how autophagosomes generated at distal axons acquire dynein motors for retrograde transport toward the soma. In this paper, we demonstrate that late endosome (LE)–loaded dynein–snapin complexes drive AV retrograde transport in axons upon fusion of autophagosomes with LEs into amphisomes. Blocking the fusion with syntaxin17 knockdown reduced recruitment of dynein motors to AVs, thus immobilizing them in axons. Deficiency in dynein–snapin coupling impaired AV transport, resulting in AV accumulation in neurites and synaptic terminals. Altogether, our study provides the first evidence that autophagosomes recruit dynein through fusion with LEs and reveals a new motor–adaptor sharing mechanism by which neurons may remove distal AVs engulfing aggregated proteins and dysfunctional organelles for efficient degradation in the soma. PMID:25940348

Discovery of a vezatin-like protein for dynein-mediated early endosome transport

PubMed Central

Yao, Xuanli; Arst, Herbert N.; Wang, Xiangfeng; Xiang, Xin

2015-01-01

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo. PMID:26378255

CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans.

PubMed

Efimov, Vladimir P; Zhang, Jun; Xiang, Xin

2006-04-01

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).

CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization in Aspergillus nidulansV⃞

PubMed Central

Efimov, Vladimir P.; Zhang, Jun; Xiang, Xin

2006-01-01

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32°C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150Glued dynactin, and NUDF were all seen as plus-end comets at 32°C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32°C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42°C). PMID:16467375

Structural features of LC8-induced self-association of swallow.

PubMed

Kidane, Ariam I; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-09-03

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Structural Features of LC8-Induced Self Association of Swallow†

PubMed Central

Kidane, Ariam I.; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-01-01

Cell function depends on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than dynein and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the mechanistic and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, inter-monomer NOEs assigned to residues expected at interface positions and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled-coil. Secondary chemical shifts and 15N backbone relaxation identify the boundaries and distinguishing structural features of the coiled-coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled-coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled-coil is critical for bicoid mRNA localization because it could induce structural organization of Swallow, which except for the central LC8-promoted coiled-coil is primarily disordered. PMID:23914803

ATM Substrate Chk2-interacting Zn2+ Finger (ASCIZ) Is a Bi-functional Transcriptional Activator and Feedback Sensor in the Regulation of Dynein Light Chain (DYNLL1) Expression*

PubMed Central

Jurado, Sabine; Conlan, Lindus A.; Baker, Emma K.; Ng, Jane-Lee; Tenis, Nora; Hoch, Nicolas C.; Gleeson, Kimberly; Smeets, Monique; Izon, David; Heierhorst, Jörg

2012-01-01

The highly conserved DYNLL1 (LC8) protein was originally discovered as a light chain of the dynein motor complex, but is increasingly emerging as a sequence-specific regulator of protein dimerization with hundreds of targets and wide-ranging cellular functions. Despite its important roles, DYNLL1's own regulation remains poorly understood. Here we identify ASCIZ (ATMIN/ZNF822), an essential Zn2+ finger protein with dual roles in the DNA base damage response and as a developmental transcription factor, as a conserved regulator of Dynll1 gene expression. DYNLL1 levels are reduced by ∼10-fold in the absence of ASCIZ in human, mouse and chicken cells. ASCIZ binds directly to the Dynll1 promoter and regulates its activity in a Zn2+ finger-dependent manner. DYNLL1 protein in turn interacts with ten binding sites in the ASCIZ transcription activation domain, and high DYNLL1 levels inhibit the transcriptional activity of ASCIZ. In addition, DYNLL1 was also required for DNA damage-induced ASCIZ focus formation. The dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. The ASCIZ-DYNLL1 feedback loop represents a novel mechanism for auto-regulation of gene expression, where the gene product directly inhibits the transcriptional activator while bound at its own promoter. PMID:22167198

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

PubMed

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-09-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-X L , recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-X L inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

A solid-state control system for dynein-based ciliary/flagellar motility

PubMed Central

2013-01-01

Ciliary and flagellar beating requires the coordinated action of multiple dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm dynein essential for waveform determination. PMID:23569213

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs

PubMed Central

Dix, Carly I.; Soundararajan, Harish Chandra; Dzhindzhev, Nikola S.; Begum, Farida; Suter, Beat; Ohkura, Hiroyuki; Stephens, Elaine

2013-01-01

Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end–directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin. PMID:23918939

Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein.

PubMed

Klinman, Eva; Holzbaur, Erika L F

2015-07-21

Axonal transport is essential for neuronal function, and defects in transport are associated with multiple neurodegenerative diseases. Aberrant cyclin-dependent kinase 5 (CDK5) activity, driven by the stress-induced activator p25, also is observed in these diseases. Here we show that elevated CDK5 activity increases the frequency of nonprocessive events for a range of organelles, including lysosomes, autophagosomes, mitochondria, and signaling endosomes. Transport disruption induced by aberrant CDK5 activation depends on the Lis1/Ndel1 complex, which directly regulates dynein activity. CDK5 phosphorylation of Ndel1 favors a high affinity Lis1/Ndel/dynein complex that blocks the ATP-dependent release of dynein from microtubules, inhibiting processive motility of dynein-driven cargo. Similar transport defects observed in neurons from a mouse model of amyotrophic lateral sclerosis are rescued by CDK5 inhibition. Together, these studies identify CDK5 as a Lis1/Ndel1-dependent regulator of transport in stressed neurons, and suggest that dysregulated CDK5 activity contributes to the transport deficits observed during neurodegeneration. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

PubMed Central

Olcese, Chiara; Patel, Mitali P.; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J.; Vaughan, Cara K.; Hayward, Jane; Goldenberg, Alice; Emes, Richard D.; Munye, Mustafa M.; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean- François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R.; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M. K.; Antonarakis, Stylianos E.; Loebinger, Michael R.; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Beales, Philip L.; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Allan, Daly; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; McCarthy, Shane; Muddyman, Dawn; Muntoni, Francesco; Parker, Victoria; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter J.; Schmidts, Miriam; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M.

2017-01-01

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins. PMID:28176794

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

PubMed

Olcese, Chiara; Patel, Mitali P; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J; Vaughan, Cara K; Hayward, Jane; Goldenberg, Alice; Emes, Richard D; Munye, Mustafa M; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean-François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M K; Antonarakis, Stylianos E; Loebinger, Michael R; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M

2017-02-08

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

Genetic analysis of the cytoplasmic dynein subunit families.

PubMed

Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic Analysis of the Cytoplasmic Dynein Subunit Families

PubMed Central

Pfister, K. Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M. C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

Proteins that interact with calgranulin B in the human colon cancer cell line HCT-116.

PubMed

Myung, Jae Kyung; Yeo, Seung-Gu; Kim, Kyung Hee; Baek, Kwang-Soo; Shin, Daye; Kim, Jong Heon; Cho, Jae Youl; Yoo, Byong Chul

2017-01-24

Calgranulin B is released from immune cells and can be internalized into colon cancer cells to prevent proliferation. The present study aimed to identify proteins that interact with calgranulin B to suppress the proliferation of colon cancer cells, and to obtain information on the underlying anti-tumor mechanism(s) of calgranulin B. Calgranulin B expression was induced in colon cancer cell line HCT-116 by infection with calgranulin B-FLAG expressing lentivirus, and it led to a significant suppression of cell proliferation. Proteins that interacted with calgranulin B were obtained by immunoprecipitation using whole homogenate of lentivirus-infected HCT-116 cells which expressing calgranulin B-FLAG, and identified using liquid chromatography-mass spectrometry/mass spectrometry analysis. A total of 454 proteins were identified that potentially interact with calgranulin B, and most identified proteins were associated with RNA processing, post-transcriptional modifications and the EIF2 signaling pathway. Direct interaction of calgranulin B with flotillin-1, dynein intermediate chain 1, and CD59 glycoprotein has been confirmed, and the molecules N-myc proto-oncogene protein, rapamycin-insensitive companion of mTOR, and myc proto-oncogene protein were shown to regulate calgranulin B-interacting proteins. Our results provide new insight and useful information to explain the possible mechanism(s) underlying the role of calgranulin B as an anti-tumor effector in colon cancer cells.

Sequential activities of Dynein, Mud and Asp in centrosome-spindle coupling maintain centrosome number upon mitosis.

PubMed

Bosveld, Floris; Ainslie, Anna; Bellaïche, Yohanns

2017-10-15

Centrosomes nucleate microtubules and are tightly coupled to the bipolar spindle to ensure genome integrity, cell division orientation and centrosome segregation. While the mechanisms of centrosome-dependent microtubule nucleation and bipolar spindle assembly have been the focus of numerous works, less is known about the mechanisms ensuring the centrosome-spindle coupling. The conserved NuMA protein (Mud in Drosophila ) is best known for its role in spindle orientation. Here, we analyzed the role of Mud and two of its interactors, Asp and Dynein, in the regulation of centrosome numbers in Drosophila epithelial cells. We found that Dynein and Mud mainly initiate centrosome-spindle coupling prior to nuclear envelope breakdown (NEB) by promoting correct centrosome positioning or separation, while Asp acts largely independently of Dynein and Mud to maintain centrosome-spindle coupling. Failure in the centrosome-spindle coupling leads to mis-segregation of the two centrosomes into one daughter cell, resulting in cells with supernumerary centrosomes during subsequent divisions. Altogether, we propose that Dynein, Mud and Asp operate sequentially during the cell cycle to ensure efficient centrosome-spindle coupling in mitosis, thereby preventing centrosome mis-segregation to maintain centrosome number. © 2017. Published by The Company of Biologists Ltd.

Nonlinear amplitude dynamics in flagellar beating

NASA Astrophysics Data System (ADS)

Oriola, David; Gadêlha, Hermes; Casademunt, Jaume

2017-03-01

The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating.

Dynactin Function in Mitotic Spindle Positioning

PubMed Central

Moore, Jeffrey K.; Li, Jun; Cooper, John A.

2008-01-01

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150Glued, dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150Glued was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast. PMID:18221362

Nonlinear amplitude dynamics in flagellar beating.

PubMed

Oriola, David; Gadêlha, Hermes; Casademunt, Jaume

2017-03-01

The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating.

Nonlinear amplitude dynamics in flagellar beating

PubMed Central

Casademunt, Jaume

2017-01-01

The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating. PMID:28405357

Steady dynein forces induce flutter instability and propagating waves in mathematical models of flagella

PubMed Central

Dutcher, S. K.

2016-01-01

Cilia and flagella are highly conserved organelles that beat rhythmically with propulsive, oscillatory waveforms. The mechanism that produces these autonomous oscillations remains a mystery. It is widely believed that dynein activity must be dynamically regulated (switched on and off, or modulated) on opposite sides of the axoneme to produce oscillations. A variety of regulation mechanisms have been proposed based on feedback from mechanical deformation to dynein force. In this paper, we show that a much simpler interaction between dynein and the passive components of the axoneme can produce coordinated, propulsive oscillations. Steady, distributed axial forces, acting in opposite directions on coupled beams in viscous fluid, lead to dynamic structural instability and oscillatory, wave-like motion. This ‘flutter’ instability is a dynamic analogue to the well-known static instability, buckling. Flutter also occurs in slender beams subjected to tangential axial loads, in aircraft wings exposed to steady air flow and in flexible pipes conveying fluid. By analysis of the flagellar equations of motion and simulation of structural models of flagella, we demonstrate that dynein does not need to switch direction or inactivate to produce autonomous, propulsive oscillations, but must simply pull steadily above a critical threshold force. PMID:27798276

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

PubMed Central

Valle-Tenney, Roger; Opazo, Tatiana; Cancino, Jorge; Goff, Stephen P.

2016-01-01

ABSTRACT During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm—a crowded environment where diffusion is slow—is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex. PMID:27194765

BicaudalD actively regulates microtubule motor activity in lipid droplet transport.

PubMed

Larsen, Kristoffer S; Xu, Jing; Cermelli, Silvia; Shu, Zhanyong; Gross, Steven P

2008-01-01

A great deal of sub-cellular organelle positioning, and essentially all minus-ended organelle transport, depends on cytoplasmic dynein, but how dynein's function is regulated is not well understood. BicD is established to play a critical role in mediating dynein function-loss of BicD results in improperly localized nuclei, mRNA particles, and a dispersed Golgi apparatus-however exactly what BicD's role is remains unknown. Nonetheless, it is widely believed that BicD may act to tether dynein to cargos. Here we use a combination of biophysical and biochemical studies to investigate BicD's role in lipid droplet transport during Drosophila embryogenesis. Functional loss of BicD impairs the embryo's ability to control the net direction of droplet transport; the developmentally controlled reversal in transport is eliminated. We find that minimal BicD expression (near-BicD(null)) decreases the average run length of both plus and minus end directed microtubule (MT) based transport. A point mutation affecting the BicD N-terminus has very similar effects on transport during cellularization (phase II), but in phase III (gastrulation) motion actually appears better than in the wild-type. In contrast to a simple static tethering model of BicD function, or a role only in initial dynein recruitment to the cargo, our data uncovers a new dynamic role for BicD in actively regulating transport. Lipid droplets move bi-directionally, and our investigations demonstrate that BicD plays a critical-and temporally changing-role in balancing the relative contributions of plus-end and minus-end motors to control the net direction of transport. Our results suggest that while BicD might contribute to recruitment of dynein to the cargo it is not absolutely required for such dynein localization, and it clearly contributes to regulation, helping activation/inactivation of the motors.

Structure of a Yeast Dyn2-Nup159 Complex and Molecular Basis for Dynein Light Chain-Nuclear Pore Interaction*

PubMed Central

Romes, Erin M.; Tripathy, Ashutosh; Slep, Kevin C.

2012-01-01

The nuclear pore complex gates nucleocytoplasmic transport through a massive, eight-fold symmetric channel capped by a nucleoplasmic basket and structurally unique, cytoplasmic fibrils whose tentacles bind and regulate asymmetric traffic. The conserved Nup82 complex, composed of Nsp1, Nup82, and Nup159, forms the unique cytoplasmic fibrils that regulate mRNA nuclear export. Although the nuclear pore complex plays a fundamental, conserved role in nuclear trafficking, structural information about the cytoplasmic fibrils is limited. Here, we investigate the structural and biochemical interactions between Saccharomyces cerevisiae Nup159 and the nucleoporin, Dyn2. We find that Dyn2 is predominantly a homodimer and binds arrayed sites on Nup159, promoting the Nup159 parallel homodimerization. We present the first structure of Dyn2, determined at 1.85 Å resolution, complexed with a Nup159 target peptide. Dyn2 resembles homologous metazoan dynein light chains, forming homodimeric composite substrate binding sites that engage two independent 10-residue target motifs, imparting a β-strand structure to each peptide via antiparallel extension of the Dyn2 core β-sandwich. Dyn2 recognizes a highly conserved QT motif while allowing sequence plasticity in the flanking residues of the peptide. Isothermal titration calorimetric analysis of the comparative binding of Dyn2 to two Nup159 target sites shows similar affinities (18 and 13 μm), but divergent thermal binding modes. Dyn2 homodimers are arrayed in the crystal lattice, likely mimicking the arrayed architecture of Dyn2 on the Nup159 multivalent binding sites. Crystallographic interdimer interactions potentially reflect a cooperative basis for Dyn2-Nup159 complex formation. Our data highlight the determinants that mediate oligomerization of the Nup82 complex and promote a directed, elongated cytoplasmic fibril architecture. PMID:22411995

Nanoparticles engineered to bind cellular motors for efficient delivery.

PubMed

Dalmau-Mena, Inmaculada; Del Pino, Pablo; Pelaz, Beatriz; Cuesta-Geijo, Miguel Ángel; Galindo, Inmaculada; Moros, María; de la Fuente, Jesús M; Alonso, Covadonga

2018-03-30

Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Computer simulation of flagellar movement VIII: coordination of dynein by local curvature control can generate helical bending waves.

PubMed

Brokaw, Charles J

2002-10-01

Computer simulations have been carried out with a model flagellum that can bend in three dimensions. A pattern of dynein activation in which regions of dynein activity propagate along each doublet, with a phase shift of approximately 1/9 wavelength between adjacent doublets, will produce a helical bending wave. This pattern can be termed "doublet metachronism." The simulations show that doublet metachronism can arise spontaneously in a model axoneme in which activation of dyneins is controlled locally by the curvature of each outer doublet microtubule. In this model, dyneins operate both as sensors of curvature and as motors. Doublet metachronism and the chirality of the resulting helical bending pattern are regulated by the angular difference between the direction of the moment and sliding produced by dyneins on a doublet and the direction of the controlling curvature for that doublet. A flagellum that is generating a helical bending wave experiences twisting moments when it moves against external viscous resistance. At high viscosities, helical bending will be significantly modified by twist unless the twist resistance is greater than previously estimated. Spontaneous doublet metachronism must be modified or overridden in order for a flagellum to generate the planar bending waves that are required for efficient propulsion of spermatozoa. Planar bending can be achieved with the three-dimensional flagellar model by appropriate specification of the direction of the controlling curvature for each doublet. However, experimental observations indicate that this "hard-wired" solution is not appropriate for real flagella. Copyright 2002 Wiley-Liss, Inc.

LRRC6 Mutation Causes Primary Ciliary Dyskinesia with Dynein Arm Defects

PubMed Central

Horani, Amjad; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Oren, Yifat S.; Kerem, Batsheva; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Brody, Steven L.; Elpeleg, Orly; Kerem, Eitan

2013-01-01

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects. PMID:23527195

Engineered tug-of-war between kinesin and dynein controls direction of microtubule transport in vivo

PubMed Central

Rezaul, Karim; Gupta, Dipika; Semenova, Irina; Ikeda, Kazuho; Kraikivski, Pavel; Yu, Ji; Cowan, Ann; Zaliapin, Ilya; Rodionov, Vladimir

2017-01-01

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo. PMID:26843027

Regulation of microtubule-based transport by MAP4

PubMed Central

Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

2014-01-01

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

PubMed Central

Rompolas, Panteleimon; Patel-King, Ramila S.

2010-01-01

Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony. PMID:20844081

Effectiveness of a dynein team in a tug of war helped by reduced load sensitivity of detachment: evidence from the study of bidirectional endosome transport in D. discoideum.

PubMed

Bhat, Deepak; Gopalakrishnan, Manoj

2012-08-01

Bidirectional cargo transport by molecular motors in cells is a complex phenomenon in which the cargo (usually a vesicle) alternately moves in retrograde and anterograde directions. In this case, teams of oppositely pulling motors (e.g., kinesin and dynein) bind to the cargo, simultaneously, and 'coordinate' their activity such that the motion consists of spells of positively and negatively directed segments, separated by pauses of varying duration. A set of recent experiments have analyzed the bidirectional motion of endosomes in the amoeba D. discoideum in detail. It was found that in between directional switches, a team of five to six dyneins stall a cargo against a stronger kinesin in a tug of war, which lasts for almost a second. As the mean detachment time of a kinesin under its stall load was also observed to be ∼1 s, we infer that the collective detachment time of the dynein assembly must also be similar. Here, we analyze this inference from a modeling perspective, using experimentally measured single-molecule parameters as inputs. We find that the commonly assumed exponential load-dependent detachment rate is inconsistent with observations, as it predicts that a five-dynein assembly will detach under its combined stall load in less than a hundredth of a second. A modified model where the load-dependent unbinding rate is assumed to saturate at stall-force level for super-stall loads gives results which are in agreement with experimental data. Our analysis suggests that the load-dependent detachment of a dynein in a team is qualitatively different at sub-stall and super-stall loads, a conclusion which is likely to have implications in other situations involving collective effects of many motors.

Alcohol-induced defects in hepatic transcytosis may be explained by impaired dynein function.

PubMed

Groebner, Jennifer L; Fernandez, David J; Tuma, Dean J; Tuma, Pamela L

2014-12-01

Alcoholic liver disease has been clinically well described, but the molecular mechanisms leading to hepatotoxicity have not been fully elucidated. Previously, we determined that microtubules are hyperacetylated and more stable in ethanol-treated WIF-B cells, VL-17A cells, liver slices, and in livers from ethanol-fed rats. From our recent studies, we believe that these modifications can explain alcohol-induced defects in microtubule motor-dependent protein trafficking including nuclear translocation of a subset of transcription factors. Since cytoplasmic dynein/dynactin is known to mediate both microtubule-dependent translocation and basolateral to apical/canalicular transcytosis, we predicted that transcytosis is impaired in ethanol-treated hepatic cells. We monitored transcytosis of three classes of newly synthesized canalicular proteins in polarized, hepatic WIF-B cells, an emerging model system for the study of liver disease. As predicted, canalicular delivery of all proteins tested was impaired in ethanol-treated cells. Unlike in control cells, transcytosing proteins were observed in discrete sub-canalicular puncta en route to the canalicular surface that aligned along acetylated microtubules. We further determined that the stalled transcytosing proteins colocalized with dynein/dynactin in treated cells. No changes in vesicle association were observed for either dynein or dynactin in ethanol-treated cells, but significantly enhanced dynein binding to microtubules was observed. From these results, we propose that enhanced dynein binding to microtubules in ethanol-treated cells leads to decreased motor processivity resulting in vesicle stalling and in impaired canalicular delivery. Our studies also importantly indicate that modulating cellular acetylation levels with clinically tolerated deacetylase agonists may be a novel therapeutic strategy for treating alcoholic liver disease.

Alcohol-induced defects in hepatic transcytosis may be explained by impaired dynein function

PubMed Central

Groebner, Jennifer L.; Fernandez, David J.; Tuma, Dean J.; Tuma, Pamela L.

2016-01-01

Alcoholic liver disease has been clinically well described, but the molecular mechanisms leading to hepatotoxicity have not been fully elucidated. Previously, we determined that microtubules are hyperacetylated and more stable in ethanol-treated WIF-B cells, VL-17A cells, liver slices, and in livers from ethanol-fed rats. From our recent studies, we believe that these modifications can explain alcohol-induced defects in microtubule motor-dependent protein trafficking including nuclear translocation of a subset of transcription factors. Since cytoplasmic dynein/dynactin is known to mediate both microtubule-dependent translocation and basolateral to apical/canalicular transcytosis, we predicted that transcytosis is impaired in ethanol-treated hepatic cells. We monitored transcytosis of three classes of newly synthesized canalicular proteins in polarized, hepatic WIF-B cells, an emerging model system for the study of liver disease. As predicted, canalicular delivery of all proteins tested was impaired in ethanol-treated cells. Unlike in control cells, transcytosing proteins were observed in discrete sub-canalicular puncta en route to the canalicular surface that aligned along acetylated microtubules. We further determined that the stalled transcytosing proteins colocalized with dynein/dynactin in treated cells. No changes in vesicle association were observed for either dynein or dynactin in ethanol-treated cells, but significantly enhanced dynein binding to micro-tubules was observed. From these results, we propose that enhanced dynein binding to microtubules in ethanol-treated cells leads to decreased motor processivity resulting in vesicle stalling and in impaired canalicular delivery. Our studies also importantly indicate that modulating cellular acetylation levels with clinically tolerated deacetylase agonists may be a novel therapeutic strategy for treating alcoholic liver disease. PMID:25148871

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

PubMed Central

Spiridonov, Ilia S.; McIntosh, J. Richard

2007-01-01

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end–directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole. PMID:17409356

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface

NASA Astrophysics Data System (ADS)

Ikuta, Junya; Kamisetty, Nagendra K.; Shintaku, Hirofumi; Kotera, Hidetoshi; Kon, Takahide; Yokokawa, Ryuji

2014-06-01

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved.

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules

NASA Astrophysics Data System (ADS)

Imai, Hiroshi; Shima, Tomohiro; Sutoh, Kazuo; Walker, Matthew L.; Knight, Peter J.; Kon, Takahide; Burgess, Stan A.

2015-09-01

Cytoplasmic dynein is a dimeric AAA+ motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA+ rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein's directionality and unusual stepping behaviour.

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface

PubMed Central

Ikuta, Junya; Kamisetty, Nagendra K.; Shintaku, Hirofumi; Kotera, Hidetoshi; Kon, Takahide; Yokokawa, Ryuji

2014-01-01

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved. PMID:24923426

RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs.

PubMed

McClintock, Mark A; Dix, Carly I; Johnson, Christopher M; McLaughlin, Stephen H; Maizels, Rory J; Hoang, Ha Thi; Bullock, Simon L

2018-06-26

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis -acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport. © 2018, McClintock et al.

Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.

PubMed

Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

2016-02-11

Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Substructure of the outer dynein arm

PubMed Central

1982-01-01

The substructure of the outer dynein arm has been analyzed in quick- frozen deep-etch replicas of Tetrahymena and Chlamydomonas axonemes. Each arm is found to be composed of five morphologically discrete components: an elliptical head; two spherical feet; a slender stalk; and an interdynein linker. The feet make contact with the A microtubule of each doublet; the stalk contacts the B microtubule; the head lies between the feet and stalk; and the linker associates each arm with its neighbor. The spatial relationships between these five components are found to be distinctly different in rigor (ATP-depleted) versus relaxed (ATP- or vanadate plus ATP-treated) axonemes, and the stalk appears to alter its affinity for the B microtubule in the relaxed state. Images of living cilia attached to Tetrahymena cells show that the relaxed configuration is adopted in vivo. We relate our observations to morphological and experimental studies reported by others and propose several models that suggest how this newly described dynein morphology may relate to dynein function. PMID:6218174

Integrated proteomics identified novel activation of dynein IC2-GR-COX-1 signaling in neurofibromatosis type I (NF1) disease model cells.

PubMed

Hirayama, Mio; Kobayashi, Daiki; Mizuguchi, Souhei; Morikawa, Takashi; Nagayama, Megumi; Midorikawa, Uichi; Wilson, Masayo M; Nambu, Akiko N; Yoshizawa, Akiyasu C; Kawano, Shin; Araki, Norie

2013-05-01

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella*

PubMed Central

Lin, Jianfeng; Tritschler, Douglas; Song, Kangkang; Barber, Cynthia F.; Cobb, Jennifer S.; Porter, Mary E.; Nicastro, Daniela

2011-01-01

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function. PMID:21700706

The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16

PubMed Central

Hou, Yuqing; Witman, George B.

2017-01-01

Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multisubunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that the IFT46 N-terminus, ODA16, and outer arm dynein interact for IFT of the latter. PMID:28701346

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

PubMed Central

Lippert, Lisa G.; Dadosh, Tali; Hadden, Jodi A.; Karnawat, Vishakha; Diroll, Benjamin T.; Murray, Christopher B.; Holzbaur, Erika L. F.; Schulten, Klaus; Reck-Peterson, Samara L.; Goldman, Yale E.

2017-01-01

The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein’s stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT. PMID:28533393

Structural insights into microtubule doublet interactions inaxonemes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Downing, Kenneth H.; Sui, Haixin

2007-06-06

Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of the axoneme. Recent structural studies of the axoneme have used cryo-electron tomography to reveal new details of the interactions among some of the multitude of proteins that form the axoneme and regulate its movement. Connections among the several sets of dyneins, in particular, suggest ways in which their actions may be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding the doublet's mechanical properties that are related to the bending of the axoneme, and has also offered insight into itsmore » potential role in the mechanism of dynein activity regulation.« less

Mutation analysis of genes within the dynactin complex in a cohort of hereditary peripheral neuropathies.

PubMed

Tey, S; Ahmad-Annuar, A; Drew, A P; Shahrizaila, N; Nicholson, G A; Kennerson, M L

2016-08-01

The cytoplasmic dynein-dynactin genes are attractive candidates for neurodegenerative disorders given their functional role in retrograde transport along neurons. The cytoplasmic dynein heavy chain (DYNC1H1) gene has been implicated in various neurodegenerative disorders, and dynactin 1 (DCTN1) genes have been implicated in a wide spectrum of disorders including motor neuron disease, Parkinson's disease, spinobulbar muscular atrophy and hereditary spastic paraplegia. However, the involvement of other dynactin genes with inherited peripheral neuropathies (IPN) namely, hereditary sensory neuropathy, hereditary motor neuropathy and Charcot-Marie-Tooth disease is under reported. We screened eight genes; DCTN1-6 and ACTR1A and ACTR1B in 136 IPN patients using whole-exome sequencing and high-resolution melt (HRM) analysis. Eight non-synonymous variants (including one novel variant) and three synonymous variants were identified. Four variants have been reported previously in other studies, however segregation analysis within family members excluded them from causing IPN in these families. No variants of disease significance were identified in this study suggesting the dynactin genes are unlikely to be a common cause of IPNs. However, with the ease of querying gene variants from exome data, these genes remain worthwhile candidates to assess unsolved IPN families for variants that may affect the function of the proteins. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella

PubMed Central

Gokhale, Avanti; Wirschell, Maureen

2009-01-01

Experimental analysis of isolated ciliary/flagellar axonemes has implicated the protein kinase casein kinase I (CK1) in regulation of dynein. To test this hypothesis, we developed a novel in vitro reconstitution approach using purified recombinant Chlamydomonas reinhardtii CK1, together with CK1-depleted axonemes from the paralyzed flagellar mutant pf17, which is defective in radial spokes and impaired in dynein-driven microtubule sliding. The CK1 inhibitors (DRB and CK1-7) and solubilization of CK1 restored microtubule sliding in pf17 axonemes, which is consistent with an inhibitory role for CK1. The phosphatase inhibitor microcystin-LR blocked rescue of microtubule sliding, indicating that the axonemal phosphatases, required for rescue, were retained in the CK1-depleted axonemes. Reconstitution of depleted axonemes with purified, recombinant CK1 restored inhibition of microtubule sliding in a DRB– and CK1-7–sensitive manner. In contrast, a purified “kinase-dead” CK1 failed to restore inhibition. These results firmly establish that an axonemal CK1 regulates dynein activity and flagellar motility. PMID:19752022

Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function

PubMed Central

Tanaka, Hiromitsu; Iguchi, Naoko; Toyama, Yoshiro; Kitamura, Kouichi; Takahashi, Tohru; Kaseda, Kazuhiro; Maekawa, Mamiko; Nishimune, Yoshitake

2004-01-01

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia. PMID:15340058

Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3

PubMed Central

Peñalva, Miguel A.; Zhang, Jun; Xiang, Xin; Pantazopoulou, Areti

2017-01-01

Hyphal tip cells of the fungus Aspergillus nidulans are useful for studying long-range intracellular traffic. Post-Golgi secretory vesicles (SVs) containing the RAB11 orthologue RabE engage myosin-5 as well as plus end– and minus end–directed microtubule motors, providing an experimental system with which to investigate the interplay between microtubule and actin motors acting on the same cargo. By exploiting the fact that depolymerization of F-actin unleashes SVs focused at the apex by myosin-5 to microtubule-dependent motors, we establish that the minus end–directed transport of SVs requires the dynein/dynactin supercomplex. This minus end–directed transport is largely unaffected by genetic ablation of the Hook complex adapting early endosomes (EEs) to dynein but absolutely requires p25 in dynactin. Thus dynein recruitment to two different membranous cargoes, namely EEs and SVs, requires p25, highlighting the importance of the dynactin pointed-end complex to scaffold cargoes. Finally, by studying the behavior of SVs and EEs in null and rigor mutants of kinesin-3 and kinesin-1 (UncA and KinA, respectively), we demonstrate that KinA is the major kinesin mediating the anterograde transport of SVs. Therefore SVs arrive at the apex of A. nidulans by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein. PMID:28209731

bop5 mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric waveforms

PubMed Central

VanderWaal, Kristyn E.; Yamamoto, Ryosuke; Wakabayashi, Ken-ichi; Fox, Laura; Kamiya, Ritsu; Dutcher, Susan K.; Bayly, Phillip V.; Sale, Winfield S.; Porter, Mary E.

2011-01-01

I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability. PMID:21697502

Equations of Interdoublet Separation during Flagella Motion Reveal Mechanisms of Wave Propagation and Instability

PubMed Central

Bayly, Philip V.; Wilson, Kate S.

2014-01-01

The motion of flagella and cilia arises from the coordinated activity of dynein motor protein molecules arrayed along microtubule doublets that span the length of axoneme (the flagellar cytoskeleton). Dynein activity causes relative sliding between the doublets, which generates propulsive bending of the flagellum. The mechanism of dynein coordination remains incompletely understood, although it has been the focus of many studies, both theoretical and experimental. In one leading hypothesis, known as the geometric clutch (GC) model, local dynein activity is thought to be controlled by interdoublet separation. The GC model has been implemented as a numerical simulation in which the behavior of a discrete set of rigid links in viscous fluid, driven by active elements, was approximated using a simplified time-marching scheme. A continuum mechanical model and associated partial differential equations of the GC model have remained lacking. Such equations would provide insight into the underlying biophysics, enable mathematical analysis of the behavior, and facilitate rigorous comparison to other models. In this article, the equations of motion for the flagellum and its doublets are derived from mechanical equilibrium principles and simple constitutive models. These equations are analyzed to reveal mechanisms of wave propagation and instability in the GC model. With parameter values in the range expected for Chlamydomonas flagella, solutions to the fully nonlinear equations closely resemble observed waveforms. These results support the ability of the GC hypothesis to explain dynein coordination in flagella and provide a mathematical foundation for comparison to other leading models. PMID:25296329

Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

PubMed

Nath, Shubhankar; Christian, Laura; Tan, Sarah Youngsun; Ki, Sanghee; Ehrlich, Lauren I R; Poenie, Martin

2016-09-15

Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively. Copyright © 2016 by The American Association of Immunologists, Inc.

Coin Tossing Explains the Activity of Opposing Microtubule Motors on Phagosomes.

PubMed

Sanghavi, Paulomi; D'Souza, Ashwin; Rai, Ashim; Rai, Arpan; Padinhatheeri, Ranjith; Mallik, Roop

2018-05-07

How the opposing activity of kinesin and dynein motors generates polarized distribution of organelles inside cells is poorly understood and hotly debated [1, 2]. Possible explanations include stochastic mechanical competition [3, 4], coordinated regulation by motor-associated proteins [5-7], mechanical activation of motors [8], and lipid-induced organization [9]. Here, we address this question by using phagocytosed latex beads to generate early phagosomes (EPs) that move bidirectionally along microtubules (MTs) in an in vitro assay [9]. Dynein/kinesin activity on individual EPs is recorded as real-time force generation of the motors against an optical trap. Activity of one class of motors frequently coincides with, or is rapidly followed by opposite motors. This leads to frequent and rapid reversals of EPs in the trap. Remarkably, the choice between dynein and kinesin can be explained by the tossing of a coin. Opposing motors therefore appear to function stochastically and independently of each other, as also confirmed by observing no effect on kinesin function when dynein is inhibited on the EPs. A simple binomial probability calculation based on the geometry of EP-microtubule contact explains the observed activity of dynein and kinesin on phagosomes. This understanding of intracellular transport in terms of a hypothetical coin, if it holds true for other cargoes, provides a conceptual framework to explain the polarized localization of organelles inside cells. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Dynein light chain regulates adaptive and innate B cell development by distinctive genetic mechanisms

PubMed Central

King, Ashleigh; Li, Lingli; Wong, David M.; Liu, Rui; Bamford, Rebecca; Strasser, Andreas

2017-01-01

Mechanistic differences in the development and function of adaptive, high-affinity antibody-producing B-2 cells and innate-like, “natural” antibody-producing B-1a cells remain poorly understood. Here we show that the multi-functional dynein light chain (DYNLL1/LC8) plays important roles in the establishment of B-1a cells in the peritoneal cavity and in the ongoing development of B-2 lymphoid cells in the bone marrow of mice. Epistasis analyses indicate that Dynll1 regulates B-1a and early B-2 cell development in a single, linear pathway with its direct transcriptional activator ASCIZ (ATMIN/ZNF822), and that the two genes also have complementary functions during late B-2 cell development. The B-2 cell defects caused by loss of DYNLL1 were associated with lower levels of the anti-apoptotic protein BCL-2, and could be supressed by deletion of pro-apoptotic BIM which is negatively regulated by both DYNLL1 and BCL-2. Defects in B cell development caused by loss of DYNLL1 could also be partially suppressed by a pre-arranged SWHEL Igm-B cell receptor transgene. In contrast to the rescue of B-2 cell numbers, the B-1a cell deficiency in Dynll1-deleted mice could not be suppressed by the loss of Bim, and was further compounded by the SWHEL transgene. Conversely, oncogenic MYC expression, which is synthetic lethal with Dynll1 deletion in B-2 cells, did not further reduce B-1a cell numbers in Dynll1-defcient mice. Finally, we found that the ASCIZ-DYNLL1 axis was also required for the early-juvenile development of aggressive MYC-driven and p53-deficient B cell lymphomas. These results identify ASCIZ and DYNLL1 as the core of a transcriptional circuit that differentially regulates the development of the B-1a and B-2 B lymphoid cell lineages and plays a critical role in lymphomagenesis. PMID:28922373

Time-Dependent Measure of a Nano-Scale Force-Pulse Driven by the Axonemal Dynein Motors in Individual Live Sperm Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, M J; Rudd, R E; McElfresh, M W

Nano-scale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces would be important to developing motile biomimetic nanodevices powered by biological motors for Nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding giving rise to rhythmic beating of the flagellum. This force-generating action makes it possible for the sperm cell to move through viscous media. Here we report new nano-scale information on how the propulsive force is generated by the sperm flagellum and how this force varies over time. Single cell recordings reveal discretemore » {approx}50 ms pulses oscillating with amplitude 9.8 {+-} 2.6 nN independent of pulse frequency (3.5-19.5 Hz). The average work carried out by each cell is 4.6 x 10{sup -16} J per pulse, equivalent to the hydrolysis of {approx}5,500 ATP molecules. The mechanochemical coupling at each active dynein head is {approx}2.2 pN/ATP, and {approx}3.9 pN per dynein arm, in agreement with previously published values obtained using different methods.« less

Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces.

PubMed

Barisic, Marin; Aguiar, Paulo; Geley, Stephan; Maiato, Helder

2014-12-01

Accurate chromosome segregation during cell division in metazoans relies on proper chromosome congression at the equator. Chromosome congression is achieved after bi-orientation to both spindle poles shortly after nuclear envelope breakdown, or by the coordinated action of motor proteins that slide misaligned chromosomes along pre-existing spindle microtubules. These proteins include the minus-end-directed kinetochore motor dynein, and the plus-end-directed motors CENP-E at kinetochores and chromokinesins on chromosome arms. However, how these opposite and spatially distinct activities are coordinated to drive chromosome congression remains unknown. Here we used RNAi, chemical inhibition, kinetochore tracking and laser microsurgery to uncover the functional hierarchy between kinetochore and arm-associated motors, exclusively required for congression of peripheral polar chromosomes in human cells. We show that dynein poleward force counteracts chromokinesins to prevent stabilization of immature/incorrect end-on kinetochore-microtubule attachments and random ejection of polar chromosomes. At the poles, CENP-E becomes dominant over dynein and chromokinesins to bias chromosome ejection towards the equator. Thus, dynein and CENP-E at kinetochores drive congression of peripheral polar chromosomes by preventing arm-ejection forces mediated by chromokinesins from working in the wrong direction.

Regulation of dynein-mediated autophagosomes trafficking by ASM in CASMCs.

PubMed

Xu, Ming; Zhang, Qiufang; Li, Pin-Lan; Nguyen, Thaison; Li, Xiang; Zhang, Yang

2016-01-01

Acid sphingomyelinase (ASM; gene symbol Smpd1) has been shown to play a crucial role in autophagy maturation by controlling lysosomal fusion with autophagosomes in coronary arterial smooth muscle cells (CASMCs). However, the underlying molecular mechanism by which ASM controls autophagolysosomal fusion remains unknown. In primary cultured CASMCs, lysosomal Ca2+ induced by 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer) was markedly attenuated by ASM deficiency or TRPML1 gene silencing suggesting that ASM signaling is required for TRPML1 channel activity and subsequent lysosomal Ca(2+) release. In these CASMCs, ASM deficiency or TRPML1 gene silencing markedly inhibited 7-Ket-induced dynein activation. In addition, 7-Ket-induced autophagosome trafficking, an event associated with lysosomal Ca(2+) release and dynein activity, was significantly inhibited in ASM-deficient (Smpd1(-/-)) CASMCs compared to that in Smpd1(+/+) CASMCs. Finally, overexpression of TRPML1 proteins restored 7-Ket-induced lysosomal Ca(2+) release and autophagosome trafficking in Smpd1-/- CASMCs. Collectively, these results suggest that ASM plays a critical role in regulating lysosomal TRPML1-Ca(2+) signaling and subsequent dynein-mediated autophagosome trafficking, which leads its role in controlling autophagy maturation in CASMCs under atherogenic stimulation.

Dynein pulling forces counteract lamin-mediated nuclear stability during nuclear envelope repair

PubMed Central

Penfield, Lauren; Wysolmerski, Brian; Mauro, Michael; Farhadifar, Reza; Martinez, Michael A.; Biggs, Ronald; Wu, Hai-Yin; Broberg, Curtis; Needleman, Daniel; Bahmanyar, Shirin

2018-01-01

Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) ruptures and repairs in interphase. The duration of NE ruptures depends on lamins; however, the underlying mechanisms and relevance to in vivo events are not known. Here, we use the Caenorhabditis elegans zygote to analyze lamin’s role in NE rupture and repair in vivo. Transient NE ruptures and subsequent NE collapse are induced by weaknesses in the nuclear lamina caused by expression of an engineered hypomorphic C. elegans lamin allele. Dynein-generated forces that position nuclei enhance the severity of transient NE ruptures and cause NE collapse. Reduction of dynein forces allows the weakened lamin network to restrict nucleo–cytoplasmic mixing and support stable NE recovery. Surprisingly, the high incidence of transient NE ruptures does not contribute to embryonic lethality, which is instead correlated with stochastic chromosome scattering resulting from premature NE collapse, suggesting that C. elegans tolerates transient losses of NE compartmentalization during early embryogenesis. In sum, we demonstrate that lamin counteracts dynein forces to promote stable NE repair and prevent catastrophic NE collapse, and thus provide the first mechanistic analysis of NE rupture and repair in an organismal context. PMID:29386297

Asymmetries in kinesin-2 and cytoplasmic dynein contributions to melanosome transport.

PubMed

De Rossi, María Cecilia; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Bruno, Luciana; Levi, Valeria

2015-09-14

The mechanisms involved in bidirectional transport along microtubules remain largely unknown. We explored the collective action of kinesin-2 and dynein motors during transport of melanosomes in Xenopus laevis melanophores. These motors are attached to organelles through accessory proteins establishing a complex molecular linker. We determined both the stiffness of this linker and the organelles speed and observed that these parameters depended on the organelle size and cargo direction. Our results suggest that melanosome transport is driven by two dissimilar teams: whereas dynein motors compete with kinesin-2 affecting the properties of plus-end directed organelles, kinesin-2 does not seem to play a similar role during minus-end transport. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load.

PubMed

Andreasson, Johan O L; Shastry, Shankar; Hancock, William O; Block, Steven M

2015-05-04

The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length, and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains and not to the neck linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mechanochemical cycle of mammalian kinesin-2 KIF3A/B under load

PubMed Central

Andreasson, Johan O.L.; Shastry, Shankar; Hancock, William O.; Block, Steven M.

2015-01-01

Summary The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains, and not to the neck-linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. PMID:25866395

Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

PubMed Central

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

2010-01-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study.

PubMed

Zhu, Jie; Burakov, Anton; Rodionov, Vladimir; Mogilner, Alex

2010-12-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.

Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

PubMed Central

Wirschell, Maureen; Yamamoto, Ryosuke; Alford, Lea; Gokhale, Avanti; Gaillard, Anne; Sale, Winfield S.

2011-01-01

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation / dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates. PMID:21513695

A Structural Basis for How Motile Cilia Beat

PubMed Central

Satir, Peter; Heuser, Thomas; Sale, Winfield S.

2014-01-01

The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke–central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation. PMID:26955066

Functional diversification of the kinesin-14 family in land plants.

PubMed

Gicking, Allison M; Swentowsky, Kyle W; Dawe, R Kelly; Qiu, Weihong

2018-05-12

In most eukaryotes, cytoplasmic dynein serves as the primary cytoskeletal motor for minus-end-directed processes along microtubules. However, land plants lack dynein, having instead a large number of kinesin-14s, which suggests that kinesin-14s may have evolved to fill the cellular niche left by dynein. In addition, land plants do not have centrosomes, but contain specialized microtubule-based structures called phragmoplasts that facilitate the formation of new cell walls following cell division. This Review aims to compile the evidence for functional diversification of kinesin-14s in land plants. Known functions include spindle morphogenesis, microtubule-based trafficking, nuclear migration, chloroplast distribution, and phragmoplast expansion. Plant kinesin-14s have also evolved direct roles in chromosome segregation in maize and novel biochemical features such as actin transport and processive motility in the homodimeric state. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

LIS1 controls mitosis and mitotic spindle organization via the LIS1–NDEL1–dynein complex

PubMed Central

Moon, Hyang Mi; Youn, Yong Ha; Pemble, Hayley; Yingling, Jessica; Wittmann, Torsten; Wynshaw-Boris, Anthony

2014-01-01

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore–microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1–dynein complex. Overexpression of NDEL1–dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1–NDEL1–dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division. PMID:24030547

Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA InterferenceD⃞V⃞

PubMed Central

Zhu, Changjun; Zhao, Jian; Bibikova, Marina; Leverson, Joel D.; Bossy-Wetzel, Ella; Fan, Jian-Bing; Abraham, Robert T.; Jiang, Wei

2005-01-01

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail. PMID:15843429

pH dependent unfolding characteristics of DLC8 dimer: Residue level details from NMR.

PubMed

Mohan, P M Krishna; Hosur, Ramakrishna V

2008-11-01

Environment dependence of folding and unfolding of a protein is central to its function. In the same vein, knowledge of pH dependence of stability and folding/unfolding is crucial for many biophysical equilibrium and kinetic studies designed to understand protein folding mechanisms. In the present study we investigated the guanidine induced unfolding transition of dynein light chain protein (DLC8), a cargo adaptor of the dynein complex in the pH range 7-10. It is observed that while the protein remains a dimer in the entire pH range, its stability is somewhat reduced at alkaline pH. Global unfolding features monitored using fluorescence spectroscopy revealed that the unfolding transition of DLC8 at pH 7 is best described by a three-state model, whereas, that at pH 10 is best described by a two-state model. Chemical shift perturbations due to pH change provided insights into the corresponding residue level structural perturbations in the DLC8 dimer. Likewise, backbone (15)N relaxation measurements threw light on the corresponding motional changes in the dimeric protein. These observations have been rationalized on the basis of expected changes with increasing pH in the protonation states of the titratable residues on the structure of the protein. These, in turn provide an explanation for the change from three-state to two-state guanidine induced unfolding transition as the pH is increased from 7 to 10. All these results exemplify and highlight the role of environment vis-à-vis the sequence and structure of a given protein in dictating its folding/unfolding characteristics.

Sliding of microtubules by a team of dynein motors: Understanding the effect of spatial distribution of motor tails and mutual exclusion of motor heads on microtubules

NASA Astrophysics Data System (ADS)

Singh, Hanumant Pratap; Takshak, Anjneya; Mall, Utkarsh; Kunwar, Ambarish

2016-06-01

Molecular motors are natural nanomachines that use the free energy released from ATP hydrolysis to generate mechanical forces. Cytoplasmic dynein motors often work collectively as a team to drive important processes such as axonal growth, proplatelet formation and mitosis, as forces generated by single motors are insufficient. A large team of dynein motors is used to slide cytoskeletal microtubules with respect to one another during the process of proplatelet formation and axonal growth. These motors attach to a cargo microtubule via their tail domains, undergo the process of detachment and reattachment of their head domains on another track microtubule, while sliding the cargo microtubule along the track. Traditional continuum/mean-field approaches used in the past are not ideal for studying the sliding mechanism of microtubules, as they ignore spatial and temporal fluctuations due to different possible distributions of motor tails on cargo filament, as well as binding/unbinding of motors from their track. Therefore, these models cannot be used to address important questions such as how the distribution of motor tails on microtubules, or how the mutual exclusion of motor heads on microtubule tracks affects the sliding velocity of cargo microtubule. To answer these, here we use a computational stochastic model where we model each dynein motor explicitly. In our model, we use both random as well as uniform distributions of dynein motors on cargo microtubule, as well as mutual exclusion of motors on microtubule tracks. We find that sliding velocities are least affected by the distribution of motor tails on microtubules, whereas they are greatly affected by mutual exclusion of motor heads on microtubule tracks. We also find that sliding velocity depends on the length of cargo microtubule if mutual exclusion among motor heads is considered.

Internal friction and mode relaxation in a simple chain model.

PubMed

Fugmann, S; Sokolov, I M

2009-12-21

We consider the equilibrium relaxation properties of the end-to-end distance and of the principal components in a one-dimensional polymer chain model with nonlinear interaction between the beads. While for the single-well potentials these properties are similar to the ones of a Rouse chain, for the double-well interaction potentials, modeling internal friction, they differ vastly from the ones of the harmonic chain at intermediate times and intermediate temperatures. This minimal description within a one-dimensional model mimics the relaxation properties found in much more complex polymer systems. Thus, the relaxation time of the end-to-end distance may grow by orders of magnitude at intermediate temperatures. The principal components (whose directions are shown to coincide with the normal modes of the harmonic chain, whatever interaction potential is assumed) not only display larger relaxation times but also subdiffusive scaling.

Dynamic allostery of protein alpha helical coiled-coils

PubMed Central

Hawkins, Rhoda J; McLeish, Tom C.B

2005-01-01

Alpha helical coiled-coils appear in many important allosteric proteins such as the dynein molecular motor and bacteria chemotaxis transmembrane receptors. As a mechanism for transmitting the information of ligand binding to a distant site across an allosteric protein, an alternative to conformational change in the mean static structure is an induced change in the pattern of the internal dynamics of the protein. We explore how ligand binding may change the intramolecular vibrational free energy of a coiled-coil, using parameterized coarse-grained models, treating the case of dynein in detail. The models predict that coupling of slide, bend and twist modes of the coiled-coil transmits an allosteric free energy of ∼2kBT, consistent with experimental results. A further prediction is a quantitative increase in the effective stiffness of the coiled-coil without any change in inherent flexibility of the individual helices. The model provides a possible and experimentally testable mechanism for transmission of information through the alpha helical coiled-coil of dynein. PMID:16849225

Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor

PubMed Central

Mizuno, Katsutoshi; Shiba, Kogiku; Okai, Masahiko; Takahashi, Yusuke; Shitaka, Yuji; Oiwa, Kazuhiro; Tanokura, Masaru; Inaba, Kazuo

2012-01-01

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca2+ influx through specific Ca2+ channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca2+ is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca2+-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca2+ concentrations. This study describes the missing link between chemoattractant-mediated Ca2+ signaling and motor-driven microtubule sliding during sperm chemotaxis. PMID:23169663

Phosphorylation of Nlp by Plk1 negatively regulates its dynein-dynactin-dependent targeting to the centrosome.

PubMed

Casenghi, Martina; Barr, Francis A; Nigg, Erich A

2005-11-01

When cells enter mitosis the microtubule (MT) network undergoes a profound rearrangement, in part due to alterations in the MT nucleating and anchoring properties of the centrosome. Ninein and the ninein-like protein (Nlp) are centrosomal proteins involved in MT organisation in interphase cells. We show that the overexpression of these two proteins induces the fragmentation of the Golgi, and causes lysosomes to disperse toward the cell periphery. The ability of Nlp and ninein to perturb the cytoplasmic distribution of these organelles depends on their ability to interact with the dynein-dynactin motor complex. Our data also indicate that dynactin is required for the targeting of Nlp and ninein to the centrosome. Furthermore, phosphorylation of Nlp by the polo-like kinase 1 (Plk1) negatively regulates its association with dynactin. These findings uncover a mechanism through which Plk1 helps to coordinate changes in MT organisation with cell cycle progression, by controlling the dynein-dynactin-dependent transport of centrosomal proteins.

Regulation of mitochondria-dynactin interaction and mitochondrial retrograde transport in axons.

PubMed

Drerup, Catherine M; Herbert, Amy L; Monk, Kelly R; Nechiporuk, Alex V

2017-04-17

Mitochondrial transport in axons is critical for neural circuit health and function. While several proteins have been found that modulate bidirectional mitochondrial motility, factors that regulate unidirectional mitochondrial transport have been harder to identify. In a genetic screen, we found a zebrafish strain in which mitochondria fail to attach to the dynein retrograde motor. This strain carries a loss-of-function mutation in actr10 , a member of the dynein-associated complex dynactin. The abnormal axon morphology and mitochondrial retrograde transport defects observed in actr10 mutants are distinct from dynein and dynactin mutant axonal phenotypes. In addition, Actr10 lacking the dynactin binding domain maintains its ability to bind mitochondria, arguing for a role for Actr10 in dynactin-mitochondria interaction. Finally, genetic interaction studies implicated Drp1 as a partner in Actr10-dependent mitochondrial retrograde transport. Together, this work identifies Actr10 as a factor necessary for dynactin-mitochondria interaction, enhancing our understanding of how mitochondria properly localize in axons.

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution*

PubMed Central

Soares, Dinesh C.; Bradshaw, Nicholas J.; Zou, Juan; Kennaway, Christopher K.; Hamilton, Russell S.; Chen, Zhuo A.; Wear, Martin A.; Blackburn, Elizabeth A.; Bramham, Janice; Böttcher, Bettina; Millar, J. Kirsty; Barlow, Paul N.; Walkinshaw, Malcolm D.; Rappsilber, Juri; Porteous, David J.

2012-01-01

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly. PMID:22843697

TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

PubMed Central

Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

2013-01-01

Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

Mutations in ZMYND10, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms in Humans and Flies, Cause Primary Ciliary Dyskinesia

PubMed Central

Moore, Daniel J.; Onoufriadis, Alexandros; Shoemark, Amelia; Simpson, Michael A.; zur Lage, Petra I.; de Castro, Sandra C.; Bartoloni, Lucia; Gallone, Giuseppe; Petridi, Stavroula; Woollard, Wesley J.; Antony, Dinu; Schmidts, Miriam; Didonna, Teresa; Makrythanasis, Periklis; Bevillard, Jeremy; Mongan, Nigel P.; Djakow, Jana; Pals, Gerard; Lucas, Jane S.; Marthin, June K.; Nielsen, Kim G.; Santoni, Federico; Guipponi, Michel; Hogg, Claire; Antonarakis, Stylianos E.; Emes, Richard D.; Chung, Eddie M.K.; Greene, Nicholas D.E.; Blouin, Jean-Louis; Jarman, Andrew P.; Mitchison, Hannah M.

2013-01-01

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects. PMID:23891471

Art Advancing Science: Filmmaking Leads to Molecular Insights at the Nanoscale.

PubMed

Reilly, Charles; Ingber, Donald E

2017-12-26

Many have recognized the potential value of facilitating activities that span the art-science interface for the benefit of society; however, there are few examples that demonstrate how pursuit of an artistic agenda can lead to scientific insights. Here, we describe how we set out to produce an entertaining short film depicting the fertilization of the egg by sperm as a parody of a preview for another Star Wars movie to excite the public about science, but ended up developing a simulation tool for multiscale modeling. To produce an aesthetic that communicates mechanical continuity across spatial scales, we developed custom strategies that integrate physics-based animation software from the entertainment industry with molecular dynamics simulation tools, using experimental data from research publications. Using this approach, we were able to depict biological physicality across multiple spatial scales, from how sperm tails move to collective molecular behavior within the axoneme to how the molecular motor, dynein, produces force at the nanometer scale. The dynein simulations, which were validated by replicating results of past simulations and cryo-electron microscopic studies, also predicted a potential mechanism for how ATP hydrolysis drives dynein motion along the microtubule as well as how dynein changes its conformation when it goes through the power stroke. Thus, pursuit of an artistic work led to insights into biology at the nanoscale as well as the development of a highly generalizable modeling and simulation technology that has utility for nanoscience and any other area of scientific investigation that involves analysis of complex multiscale systems.

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

PubMed Central

Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

2014-01-01

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

KCH kinesin drives nuclear transport and cytoskeletal coalescence for tip cell growth.

PubMed

Yamada, Moé; Goshima, Gohta

2018-06-07

Long-distance transport along microtubules (MTs) is critical for intracellular organisation. In animals, antagonistic motor proteins kinesin (plus end-directed) and dynein (minus end-directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are absent in plant genomes. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH (kinesin with calponin homology domain)-which can also bind actin-drives MT minus end-directed nuclear transport in the moss Physcomitrella patens. When all four KCH genes were deleted, the nucleus was not maintained in the cell centre, but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localized to MTs, including at the MT focal point near the tip of protonemal cells, where MT plus ends coalesced with actin filaments. MT focus was not stably maintained in KCH KO lines, whereas actin destabilisation also disrupted the MT focus in wild-type lines despite KCH remaining on unfocused MTs. KCH had distinct functions in nuclear transport and tip growth, as a truncated KCH construct restored nuclear transport activity, but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a MT crosslinker, reminiscent of the versatile animal dynein. © 2018 American Society of Plant Biologists. All rights reserved.

Importance of anisotropy in detachment rates for force production and cargo transport by a team of motor proteins.

PubMed

Takshak, Anjneya; Kunwar, Ambarish

2016-05-01

Many cellular processes are driven by collective forces generated by a team consisting of multiple molecular motor proteins. One aspect that has received less attention is the detachment rate of molecular motors under mechanical force/load. While detachment rate of kinesin motors measured under backward force increases rapidly for forces beyond stall-force; this scenario is just reversed for non-yeast dynein motors where detachment rate from microtubule decreases, exhibiting a catch-bond type behavior. It has been shown recently that yeast dynein responds anisotropically to applied load, i.e. detachment rates are different under forward and backward pulling. Here, we use computational modeling to show that these anisotropic detachment rates might help yeast dynein motors to improve their collective force generation in the absence of catch-bond behavior. We further show that the travel distance of cargos would be longer if detachment rates are anisotropic. Our results suggest that anisotropic detachment rates could be an alternative strategy for motors to improve the transport properties and force production by the team. © 2016 The Protein Society.

Golgi Positioning

PubMed Central

Yadav, Smita; Linstedt, Adam D.

2011-01-01

The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig. 1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination. PMID:21504874

Impaired retrograde transport of axonal autophagosomes contributes to autophagic stress in Alzheimer’s disease neurons

PubMed Central

Tammineni, Prasad; Ye, Xuan; Feng, Tuancheng; Aikal, Daniyal; Cai, Qian

2017-01-01

Neurons face unique challenges of transporting nascent autophagic vacuoles (AVs) from distal axons toward the soma, where mature lysosomes are mainly located. Autophagy defects have been linked to Alzheimer’s disease (AD). However, the mechanisms underlying altered autophagy remain unknown. Here, we demonstrate that defective retrograde transport contributes to autophagic stress in AD axons. Amphisomes predominantly accumulate at axonal terminals of mutant hAPP mice and AD patient brains. Amyloid-β (Aβ) oligomers associate with AVs in AD axons and interact with dynein motors. This interaction impairs dynein recruitment to amphisomes through competitive interruption of dynein-Snapin motor-adaptor coupling, thus immobilizing them in distal axons. Consistently, deletion of Snapin in mice causes AD-like axonal autophagic stress, whereas overexpressing Snapin in hAPP neurons reduces autophagic accumulation at presynaptic terminals by enhancing AV retrograde transport. Altogether, our study provides new mechanistic insight into AD-associated autophagic stress, thus establishing a foundation for ameliorating axonal pathology in AD. DOI: http://dx.doi.org/10.7554/eLife.21776.001 PMID:28085665

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

Genome-wide association study reveals sex-specific selection signals against autosomal nucleotide variants.

PubMed

Ryu, Dongchan; Ryu, Jihye; Lee, Chaeyoung

2016-05-01

A genome-wide association study (GWAS) was conducted to examine genetic associations of common autosomal nucleotide variants with sex in a Korean population with 4183 males and 4659 females. Nine genetic association signals were identified in four intragenic and five intergenic regions (P<5 × 10(-8)). Further analysis with an independent data set confirmed two intragenic association signals in the genes encoding protein phosphatase 1, regulatory subunit 12B (PPP1R12B, intron 12, rs1819043) and dynein, axonemal, heavy chain 11 (DNAH11, intron 61, rs10255013), which are directly involved in the reproductive system. This study revealed autosomal genetic variants associated with sex ratio by GWAS for the first time. This implies that genetic variants in proximity to the association signals may influence sex-specific selection and contribute to sex ratio variation. Further studies are required to reveal the mechanisms underlying sex-specific selection.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

PubMed

Milograna, Sarah Ribeiro; Ribeiro, Márcia Regina; Baqui, Munira Muhammad Abdel; McNamara, John Campbell

2014-12-01

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²⁺/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells. Copyright © 2014 Elsevier Inc. All rights reserved.

An Essential Postdevelopmental Role for Lis1 in Mice

PubMed Central

Hines, Timothy J.; Gao, Xu; Sahu, Subhshri; Lange, Meghann M.; Turner, Jill R.

2018-01-01

LIS1 mutations cause lissencephaly (LIS), a severe developmental brain malformation. Much less is known about its role in the mature nervous system. LIS1 regulates the microtubule motor cytoplasmic dynein 1 (dynein), and as LIS1 and dynein are both expressed in the adult nervous system, Lis1 could potentially regulate dynein-dependent processes such as axonal transport. We therefore knocked out Lis1 in adult mice using tamoxifen-induced, Cre-ER-mediated recombination. When an actin promoter was used to drive Cre-ER expression (Act-Cre-ER), heterozygous Lis1 knockout (KO) caused no obvious change in viability or behavior, despite evidence of widespread recombination by a Cre reporter three weeks after tamoxifen exposure. In contrast, homozygous Lis1 KO caused the rapid onset of neurological symptoms in both male and female mice. One tamoxifen-dosing regimen caused prominent recombination in the midbrain/hindbrain, PNS, and cardiac/skeletal muscle within a week; these mice developed severe symptoms in that time frame and were killed. A different tamoxifen regimen resulted in delayed recombination in midbrain/hindbrain, but not in other tissues, and also delayed the onset of symptoms. This indicates that Lis1 loss in the midbrain/hindbrain causes the severe phenotype. In support of this, brainstem regions known to house cardiorespiratory centers showed signs of axonal dysfunction in KO animals. Transport defects, neurofilament (NF) alterations, and varicosities were observed in axons in cultured DRG neurons from KO animals. Because no symptoms were observed when a cardiac specific Cre-ER promoter was used, we propose a vital role for Lis1 in autonomic neurons and implicate defective axonal transport in the KO phenotype. PMID:29404402

Greatly Suppressed Shuttle Effect for Improved Lithium Sulfur Battery Performance through Short Chain Intermediates.

PubMed

Xu, Na; Qian, Tao; Liu, Xuejun; Liu, Jie; Chen, Yu; Yan, Chenglin

2017-01-11

The high solubility of long-chain lithium polysulfides and their infamous shuttle effect in lithium sulfur battery lead to rapid capacity fading along with low Coulombic efficiency. To address above issues, we propose a new strategy to suppress the shuttle effect for greatly enhanced lithium sulfur battery performance mainly through the formation of short-chain intermediates during discharging, which allows significant improvements including high capacity retention of 1022 mAh/g with 87% retention for 450 cycles. Without LiNO 3 -containing electrolytes, the excellent Coulombic efficiency of ∼99.5% for more than 500 cycles is obtained, suggesting the greatly suppressed shuttle effect. In situ UV/vis analysis of electrolyte during cycling reveals that the short-chain Li 2 S 2 and Li 2 S 3 polysulfides are detected as main intermediates, which are theoretically verified by density functional theory (DFT) calculations. Our strategy may open up a new avenue for practical application of lithium sulfur battery.

Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron.

PubMed

Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

2016-01-01

Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons.

Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans

PubMed Central

Crowder, Marina E.; Flynn, Jonathan R.; McNally, Karen P.; Cortes, Daniel B.; Price, Kari L.; Kuehnert, Paul A.; Panzica, Michelle T.; Andaya, Armann; Leary, Julie A.; McNally, Francis J.

2015-01-01

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin. PMID:26133383

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility

PubMed Central

Zhao, Lu; Yuan, Shiaulou; Cao, Ying; Kallakuri, Sowjanya; Li, Yuanyuan; Kishimoto, Norihito; DiBella, Linda; Sun, Zhaoxia

2013-01-01

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptinhi2394 mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptinhi2394 mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent. PMID:23858445

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility.

PubMed

Zhao, Lu; Yuan, Shiaulou; Cao, Ying; Kallakuri, Sowjanya; Li, Yuanyuan; Kishimoto, Norihito; DiBella, Linda; Sun, Zhaoxia

2013-07-30

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptin(hi2394) mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptin(hi2394) mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent.

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation

PubMed Central

Neufeld, Thomas P.

2017-01-01

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration. PMID:28100687

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus.

PubMed

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-06-09

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH.

Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus

PubMed Central

Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

2016-01-01

Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH. PMID:27277140

NMR comparison of the native energy landscapes of DLC8 dimer and monomer.

PubMed

Krishna Mohan, P M; Barve, Maneesha; Chatterjee, Amarnath; Ghosh-Roy, Anindya; Hosur, Ramakrishna V

2008-04-01

Characterization of the low energy excited states on the energy landscape of a protein is one of the exciting and challenging problems in structural biology today. In this context, we present here residue level NMR description of the low energy excited states representing locally different alternative conformations in the dynein light chain protein, in its dimeric as well as monomeric forms. Important differences have been observed between the two cases and these are not necessarily restricted to the dimer interface. Simulations indicate that the low energy excited states are within a free energy of 2-3 kcal/mol above the native state. In both the monomer and the dimer the energy landscape is very sensitive to small pH perturbations. Nearly 25% of the residues (total of residues at pH 3.0 and 3.5 for the monomer, and at pH 7.0 and 6.0 for the dimer) access alternative conformations. The observations have been rationalized on the basis of protonation-deprotonation equilibria in the side chains; histidines in the case of the dimer and aspartates/glutamates in the case of the monomer. The possible relationship of the observed ruggedness of the native energy landscape with the protein structure, and its implications to protein adaptability and unfolding have been discussed.

Photolysis of nonylphenol ethoxylates: the determination of the degradation kinetics and the intermediate products.

PubMed

Chen, Ling; Zhou, Hai-Yun; Deng, Qin-Ying

2007-06-01

The photolysis of nonylphenol ethoxylates with an average oligomers length of ten ethoxylate units (NPEO(10)) in aqueous solution under UV, as well as the influence of humic acid (HA) on the photolysis was studied. A 125W high-pressure mercury lamp was employed as the light source. The intermediate products from the photolysis were determined by LC-MS. The results indicated that NPEO(10) underwent direct photolysis upon exposed to UV. The degradation pathway was complex. Besides the generally proposed degradation pathway of ethylene oxide (EO) side chains shortening, the oxidation of alkyl chain and EO chain led to intermediates having both a carboxylated (as well as carbonylated) ethoxylate and alkyl chain of varying lengths. The hydrogenation of benzene ring was also detected. The kinetics data showed that the first order reaction kinetics could be well used to describe the kinetics of NPEO(10) degradation. In the presence of dissolved organic matter by HA addition, the performance of NPEO(10) photodegradation was reduced. The photolysis rate decreased with increased HA concentration.

Localization of the kinesin adaptor proteins trafficking kinesin proteins 1 and 2 in primary cultures of hippocampal pyramidal and cortical neurons.

PubMed

Loss, Omar; Stephenson, F Anne

2015-07-01

Neuronal function requires regulated anterograde and retrograde trafficking of mitochondria along microtubules by using the molecular motors kinesin and dynein. Previous work has established that trafficking kinesin proteins (TRAKs),TRAK1 and TRAK2, are kinesin adaptor proteins that link mitochondria to kinesin motor proteins via an acceptor protein in the mitochondrial outer membrane, etc. the Rho GTPase Miro. Recent studies have shown that TRAK1 preferentially controls mitochondrial transport in axons of hippocampal neurons by virtue of its binding to both kinesin and dynein motor proteins, whereas TRAK2 controls mitochondrial transport in dendrites resulting from its binding to dynein. This study further investigates the subcellular localization of TRAK1 and TRAK2 in primary cultures of hippocampal and cortical neurons by using both commercial antibodies and anti-TRAK1 and anti-TRAK2 antibodies raised in our own laboratory (in-house). Whereas TRAK1 was prevalently localized in axons of hippocampal and cortical neurons, TRAK2 was more prevalent in dendrites of hippocampal neurons. In cortical neurons, TRAK2 was equally distributed between axons and dendrites. Some qualitative differences were observed between commercial and in-house-generated antibody immunostaining. © 2015 Wiley Periodicals, Inc.

DRC2/CCDC65 is a central hub for assembly of the nexin–dynein regulatory complex and other regulators of ciliary and flagellar motility

PubMed Central

Bower, Raqual; Tritschler, Douglas; Mills, Kristyn VanderWaal; Heuser, Thomas; Nicastro, Daniela; Porter, Mary E.

2018-01-01

The nexin–dynein regulatory complex (N-DRC) plays a central role in the regulation of ciliary and flagellar motility. In most species, the N-DRC contains at least 11 subunits, but the specific function of each subunit is unknown. Mutations in three subunits (DRC1, DRC2/CCDC65, DRC4/GAS8) have been linked to defects in ciliary motility in humans and lead to a ciliopathy known as primary ciliary dyskinesia (PCD). Here we characterize the biochemical, structural, and motility phenotypes of two mutations in the DRC2 gene of Chlamydomonas. Using high-resolution proteomic and structural approaches, we find that the C-terminal region of DRC2 is critical for the coassembly of DRC2 and DRC1 to form the base plate of N-DRC and its attachment to the outer doublet microtubule. Loss of DRC2 in drc2 mutants disrupts the assembly of several other N-DRC subunits and also destabilizes the assembly of several closely associated structures such as the inner dynein arms, the radial spokes, and the calmodulin- and spoke-associated complex. Our study provides new insights into the range of ciliary defects that can lead to PCD. PMID:29167384

NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

PubMed Central

Seldin, Lindsey; Muroyama, Andrew; Lechler, Terry

2016-01-01

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures. DOI: http://dx.doi.org/10.7554/eLife.12504.001 PMID:26765568

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

PubMed Central

Dewey, Evan B.; Johnston, Christopher A.

2017-01-01

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. PMID:28747439

Thermal drift is enough to drive a single microtubule along its axis even in the absence of motor proteins.

PubMed Central

Nakata, T; Sato-Yoshitake, R; Okada, Y; Noda, Y; Hirokawa, N

1993-01-01

One-dimensional diffusion of microtubules (MTs), a back-and-forth motion of MTs due to thermal diffusion, was reported in dynein motility assay. The interaction between MTs and dynein that allows such motion was implicated in its importance in the force generating cycle of dynein ATPase cycle. However, it was not known whether the phenomenon is special to motor proteins. Here we show two independent examples of one-dimensional diffusion of MTs in the absence of motor proteins. Dynamin, a MT-activated GTPase, causes a nucleotide dependent back-and-forth movement of single MT up to 1 micron along the longitudinal axes, although the MT never showed unidirectional consistent movement. Quantitative analysis of the motion and its nucleotide condition indicates that the motion is due to a thermal driven diffusion, restricted to one dimension, under the weak interaction between MT and dynamin. However, specific protein-protein interaction is not essential for the motion, because similar back-and-forth movement of MT was achieved on coverslips coated with only 0.8% methylcellulose. Both cases demonstrate that thermal diffusion could provide a considerable sliding of MTs only if MTs are restricted on the surface appropriately. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:7906153

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

PubMed Central

Horani, Amjad; Brody, Steven L.; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Ta-shma, Asaf; Wilson, Kate S.; Bayly, Philip V.; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Elpeleg, Orly; Kerem, Eitan

2013-01-01

Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis. PMID:23991085

Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

PubMed Central

Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia

2015-01-01

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise

PubMed Central

Wakabayashi, Ken-ichi; King, Stephen M.

2006-01-01

Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+-binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility. PMID:16754958

Remodeling of tick cytoskeleton in response to infection with Anaplasma phagocytophilum.

PubMed

Cabezas-Cruz, Alejandro; Alberdi, Pilar; Valdes, James J; Villar, Margarita; de la Fuente, Jose

2017-06-01

The obligate intracellular pathogen Anaplasma phagocytophilum infects vertebrate and tick hosts. In this study, a genome-wide search for cytoskeleton components was performed in the tick vector, Ixodes scapularis . The available transcriptomics and proteomics data was then used to characterize the mRNA and protein levels of I. scapularis cytoskeleton components in response to A. phagocytophilum infection. The results showed that cytoskeleton components described in other model organisms were present in the I. scapularis genome. One type of intermediate filaments (lamin), a family of septins that was recently implicated in the cellular response to intracellular pathogens, and several members of motor proteins (kinesins and dyneins) that could be implicated in the cytoplasmic movements of A. phagocytophilum were found. The results showed that levels of tubulin, actin, septin, actin-related proteins and motor proteins were affected by A. phagocytophilum , probably to facilitate infection in I. scapularis . Functional studies demonstrated a role for selected cytoskeleton components in pathogen infection. These results provided a more comprehensive view of the cytoskeletal components involved in the response to A. phagocytophilum infection in ticks.

Phenylalanine 445 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences C-Ring cyclization and deprotonation reactions.

PubMed

Wu, Tung-Kung; Liu, Yuan-Ting; Chiu, Feng-Hsuan; Chang, Cheng-Hsiang

2006-10-12

[reaction: see text] We describe the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation-pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.

Precise low cost chain gears for heliostats

NASA Astrophysics Data System (ADS)

Liedke, Phillip; Lewandowski, Arkadiusz; Pfahl, Andreas; Hölle, Erwin

2016-05-01

This work investigates the potential of chain gears as precise and low cost driving systems for rim drive heliostats. After explaining chain gear basics the polygon effect and chain lengthening are investigated. The polygon effect could be measured by a heliostat with chain rim gear and the chain lengthening with an accordant test set up. Two gear stages are scope of this work: a rim gear and an intermediate gear. Dimensioning, pretensioning and designing for both stages are explained.

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

PubMed Central

Gable, Alyssa; Qiu, Minhua; Titus, Janel; Balchand, Sai; Ferenz, Nick P.; Ma, Nan; Collins, Elizabeth S.; Fagerstrom, Carey; Ross, Jennifer L.; Yang, Ge; Wadsworth, Patricia

2012-01-01

Kinesin-5 is an essential mitotic motor. However, how its spatial–temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification–tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end–directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end–directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2. PMID:22337772

Nuclear movement in fungi.

PubMed

Xiang, Xin

2017-12-11

Nuclear movement within a cell occurs in a variety of eukaryotic organisms including yeasts and filamentous fungi. Fungal molecular genetic studies identified the minus-end-directed microtubule motor cytoplasmic dynein as a critical protein for nuclear movement or orientation of the mitotic spindle contained in the nucleus. Studies in the budding yeast first indicated that dynein anchored at the cortex via its anchoring protein Num1 exerts pulling force on an astral microtubule to orient the anaphase spindle across the mother-daughter axis before nuclear division. Prior to anaphase, myosin V interacts with the plus end of an astral microtubule via Kar9-Bim1/EB1 and pulls the plus end along the actin cables to move the nucleus/spindle close to the bud neck. In addition, pushing or pulling forces generated from cortex-linked polymerization or depolymerization of microtubules drive nuclear movements in yeasts and possibly also in filamentous fungi. In filamentous fungi, multiple nuclei within a hyphal segment undergo dynein-dependent back-and-forth movements and their positioning is also influenced by cytoplasmic streaming toward the hyphal tip. In addition, nuclear movement occurs at various stages of fungal development and fungal infection of plant tissues. This review discusses our current understanding on the mechanisms of nuclear movement in fungal organisms, the importance of nuclear positioning and the regulatory strategies that ensure the proper positioning of nucleus/spindle. Published by Elsevier Ltd.

A quantitative examination of the role of cargo-exerted forces in axonal transport

PubMed Central

Mitchell, Cassie S.; Lee, Robert H.

2009-01-01

Axonal transport, via molecular motors kinesin and dynein, is a critical process in supplying the necessary constituents to maintain normal neuronal function. In this study, we predict the role of cooperativity by motors of the same polarity across the entire spectrum of physiological axonal transport. That is, we examined how the number of motors, either kinesin or dynein, working together to move a cargo, results in the experimentally determined velocity profiles seen in fast and slow anterograde and retrograde transport. We quantified the physiological forces exerted on a motor by a cargo as a function of cargo size, transport velocity, and transport type. Our results show that the force exerted by our base case neurofilament (DNF=10nm, LNF=1.6μm) is ~1.25pN at 600nm/s; additionally, the force exerted by our base case organelle (DOrg=1μm) at 1,000nm/s is ~5.7pN. Our results indicate that while a single motor can independently carry an average cargo, cooperativity is required to produce the experimental velocity profiles for fast transport. However, no cooperativity is required to produce the slow transport velocity profiles; thus, a single dynein or kinesin can carry the average neurofilament retrogradely or anterogradely, respectively. The potential role cooperativity may play in the hypothesized mechanisms of motoneuron transport diseases such as Amyotrophic Lateral Sclerosis (ALS) is discussed. PMID:19150364

Metabolic reconstructions identify plant 3-methylglutaconyl-CoA hydratase that is crucial for branched-chain amino acid catabolism in mitochondria

USDA-ARS?s Scientific Manuscript database

The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are essential nutrients for mammals. In plants, they double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates...

From labdanes to drimanes. Degradation of the side chain of dihydrozamoranic acid.

PubMed

Rodilla, Jesús M L; Díez, D; Urones, J G; Rocha, Pedro M

2004-04-30

A new route for the degradation of the saturated side chain of dihydrozamoranic acid has been devised, giving an advanced intermediate, compound 14, useful for the synthesis of insect antifeedants such as warburganal and polygodial.

Evaluation of the Molecular Structural Parameters of Normal Rice Starch and Their Relationships with Its Thermal and Digestion Properties.

PubMed

Lin, Lingshang; Zhang, Qing; Zhang, Long; Wei, Cunxu

2017-09-12

The molecular structural parameters of six normal rice starches with different amylose contents were investigated through their iodine absorption spectra and gel permeation chromatography of fully branched and debranched starches. The thermal and digestion properties of starches were also determined and their relationships with molecular structural parameters were analyzed. Results showed that the molecular structural parameters of maximum absorption wavelength, blue value (BV), optical density 620 nm/550 nm (OD 620/550), amylose, intermediate component, and amylopectin, including its short branch-chains, long branch-chains, and branching degree, had high correlation in different determining methods. The intermediate component of starch was significantly positively related to amylose and negatively related to amylopectin, and the amylopectin branching degree was significantly positively related to amylopectin content and negatively related to amylose content. The gelatinization temperatures and enthalpy of native starch were significantly positively related to BV, OD 620/550, and amylose content and negatively related to amylopectin short branch-chains. The gelatinization temperatures and enthalpy of retrograded starch were significantly negatively related to amylopectin branching degree. The digestions of gelatinized and retrograded starches were significantly negatively related to the BV, OD 620/550, amylose, and intermediate component and positively related to amylopectin and its short branch-chains and branching degree.

Mechanism of insulin fibrillation: the structure of insulin under amyloidogenic conditions resembles a protein-folding intermediate.

PubMed

Hua, Qing-xin; Weiss, Michael A

2004-05-14

Insulin undergoes aggregation-coupled misfolding to form a cross-beta assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 degrees C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal alpha-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining alpha-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain alpha-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive alpha --> beta transition characteristic of mature fibrils.

Quantifying Ciliary Dynamics during Assembly Reveals Step-wise Waveform Maturation in Airway Cells.

PubMed

Oltean, Alina; Schaffer, Andrew J; Bayly, Philip V; Brody, Steven L

2018-05-31

Motile cilia are essential for clearance of particulates and pathogens from airways. For effective transport, ciliary motor proteins and axonemal structures interact to generate the rhythmic, propulsive bending, but the mechanisms that produce a dynamic waveform remain incompletely understood. Biomechanical measures of human cilia motion and their relationships to cilia assembly are needed to illuminate the biophysics of normal cilia function, and to quantify dysfunction in ciliopathies. To these ends, we analyzed cilia motion from high-speed video microscopy of ciliated cells sampled from human lung airways compared to primary-culture cells that undergo ciliogenesis in vitro. Quantitative assessment of waveform parameters showed variations in waveform shape between individual cilia; however, general trends in waveform parameters emerged, associated with progression of cilia length and stage of differentiation. When cilia emerged from cultured cells, beat frequency was initially elevated, then fell and remained stable as cilia lengthened. In contrast, the average bending amplitude and the ability to generate force gradually increased and eventually approached values observed in ex vivo samples. Dynein arm motor proteins DNAH5, DNAH9, DNAH11, and DNAH6 were localized within specific regions of the axoneme in the ex vivo cells; however distinct stages of in vitro waveform development identified by biomechanical features were associated with the progressive movement of dyneins to the appropriate proximal or distal sections of the cilium. These observations suggest that the step-wise variation in waveform development during ciliogenesis is dependent on cilia length and potentially outer dynein arm assembly.

Kinesin and Dynein Mechanics: Measurement Methods and Research Applications.

PubMed

Abraham, Zachary; Hawley, Emma; Hayosh, Daniel; Webster-Wood, Victoria A; Akkus, Ozan

2018-02-01

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical to researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research toward novel treatment and nano-engineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will (a) discuss such techniques, specifically microscopy, atomic force microscopy (AFM), optical trapping, and magnetic tweezers, and (b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity, and dependence on adenosine triphosophate (ATP) concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

Desmin: molecular interactions and putative functions of the muscle intermediate filament protein.

PubMed

Costa, M L; Escaleira, R; Cataldo, A; Oliveira, F; Mermelstein, C S

2004-12-01

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium.

PubMed

Kutomi, Osamu; Seki, Makoto; Nakamura, Shogo; Kamachi, Hiroyuki; Noguchi, Munenori

2013-10-01

Intracellular Ca(2+) induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca(2+) signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO(3), which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca(2+)-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca(2+)-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca(2+)-induced ciliary reversal.

Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

PubMed

Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

2010-11-01

In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

PubMed Central

RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

2007-01-01

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

Effect of short-chain branching on interfacial polymer structure and dynamics under shear flow.

PubMed

Jeong, Sohdam; Kim, Jun Mo; Cho, Soowon; Baig, Chunggi

2017-11-22

We present a detailed analysis on the effect of short-chain branches on the structure and dynamics of interfacial chains using atomistic nonequilibrium molecular dynamics simulations of confined polyethylene melts in a wide range of shear rates. The intrinsically fast random motions of the short branches constantly disturb the overall chain conformation, leading to a more compact and less deformed chain structure of the short-chain branched (SCB) polymer against the imposed flow field in comparison with the corresponding linear polymer. Moreover, such highly mobile short branches along the backbone of the SCB polymer lead to relatively weaker out-of-plane wagging dynamics of interfacial chains, with highly curvy backbone structures in the intermediate flow regime. In conjunction with the contribution of short branches (as opposed to that of the backbone) to the total interfacial friction between the chains and the wall, the SCB polymer shows a nearly constant behavior in the degree of slip (d s ) with respect to shear rate in the weak-to-intermediate flow regimes. On the contrary, in the strong flow regime where irregular chain rotation and tumbling dynamics occur via intensive dynamical collisions between interfacial chains and the wall, an enhancement effect on the chain detachment from the wall, caused by short branches, leads to a steeper increase in d s for the SCB polymer than for the linear polymer. Remarkably, the SCB chains at the interface exhibit two distinct types of rolling mechanisms along the backbone, with a half-dumbbell mesoscopic structure at strong flow fields, in addition to the typical hairpin-like tumbling behavior displayed by the linear chains.

HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

PubMed Central

Jiang, Jihong; Kuan, Chia-Wei; Fotovati, Abbas; Chu, Tony LH; He, Zhengcheng; Lengyell, Tess C; Li, Huaibiao; Kroll, Torsten; Li, Amanda M; Goldowitz, Daniel; Frappart, Lucien; Ploubidou, Aspasia; Patel, Millan S; Pilarski, Linda M; Simpson, Elizabeth M; Lange, Philipp F; Allan, Douglas W

2017-01-01

Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of Hmmr-knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development. PMID:28994651

Single-molecule imaging of cytoplasmic dynein in vivo.

PubMed

Ananthanarayanan, Vaishnavi; Tolić, Iva M

2015-01-01

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytoskeletal self-organization in neuromorphogenesis.

PubMed

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge.

bicoid mRNA localises to the Drosophila oocyte anterior by random Dynein-mediated transport and anchoring

PubMed Central

Trovisco, Vítor; Belaya, Katsiaryna; Nashchekin, Dmitry; Irion, Uwe; Sirinakis, George; Butler, Richard; Lee, Jack J; Gavis, Elizabeth R; St Johnston, Daniel

2016-01-01

bicoid mRNA localises to the Drosophila oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that bicoid mRNA particles undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore, bicoid mRNA localises normally in shot2A2, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus, bicoid mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that bicoid mRNA forms 110–120 nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring. DOI: http://dx.doi.org/10.7554/eLife.17537.001 PMID:27791980

Cytoskeletal self-organization in neuromorphogenesis

PubMed Central

Dehmelt, Leif

2014-01-01

Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge. PMID:24847718

Integrated pathway analysis of nasopharyngeal carcinoma implicates the axonemal dynein complex in the Malaysian cohort.

PubMed

Chin, Yoon-Ming; Tan, Lu Ping; Abdul Aziz, Norazlin; Mushiroda, Taisei; Kubo, Michiaki; Mohd Kornain, Noor Kaslina; Tan, Geok Wee; Khoo, Alan Soo-Beng; Krishnan, Gopala; Pua, Kin-Choo; Yap, Yoke-Yeow; Teo, Soo-Hwang; Lim, Paul Vey-Hong; Nakamura, Yusuke; Lum, Chee Lun; Ng, Ching-Ching

2016-10-15

Nasopharyngeal carcinoma (NPC) is an epithelial squamous cell carcinoma on the mucosal lining of the nasopharynx. The etiology of NPC remains elusive despite many reported studies. Most studies employ a single platform approach, neglecting the cumulative influence of both the genome and transcriptome toward NPC development. We aim to employ an integrated pathway approach to identify dysregulated pathways linked to NPC. Our approach combines imputation NPC GWAS data from a Malaysian cohort as well as published expression data GSE12452 from both NPC and non-NPC nasopharynx tissues. Pathway association for GWAS data was performed using MAGENTA while for expression data, GSA-SNP was used with gene p values derived from differential expression values from GEO2R. Our study identified NPC association in the gene ontology (GO) axonemal dynein complex pathway (pGWAS-GSEA  = 1.98 × 10(-2) ; pExpr-GSEA  = 1.27 × 10(-24) ; pBonf-Combined  = 4.15 × 10(-21) ). This association was replicated in a separate cohort using gene expression data from NPC and non-NPC nasopharynx tissues (pAmpliSeq-GSEA  = 6.56 × 10(-4) ). Loss of function in the axonemal dynein complex causes impaired cilia function, leading to poor mucociliary clearance and subsequently upper or lower respiratory tract infection, the former of which includes the nasopharynx. Our approach illustrates the potential use of integrated pathway analysis in detecting gene sets involved in the development of NPC in the Malaysian cohort. © 2016 UICC.

Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terawaki, Shin-ichi, E-mail: terawaki@gunma-u.ac.jp; SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148; Yoshikane, Asuka

Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicDmore » CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site.« less

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

PubMed

Dewey, Evan B; Johnston, Christopher A

2017-09-15

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. © 2017 Dewey and Johnston. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Use of stable carbon and nitrogen isotopes to trace the larval striped bass food chain in the Sacramento-San Joaquin Estuary, California, April to September 1985

USGS Publications Warehouse

Rast, Walter; Sutton, J.E.

1989-01-01

To assess one potential cause for the decline of the striped bass fishery in the Sacramento-San Joaquin Estuary, stable carbon and nitrogen isotope ratios were used to examine the trophic structures of the larval striped bass food chain, and to trace the flux of these elements through the food chain components. Study results generally confirm a food chain consisting of the elements, phytoplankton/detritus-->zooplankton/Neomysis shrimp-->larval striped bass. The stable isotope ratios generally become more positive as one progresses from the lower to the higher trophic level food chain components, and no unusual trophic structure was found in the food chain. However, the data indicate an unidentified consumer organism occupying an intermediate position between the lower and higher trophic levels of the larval striped bass food chain. Based on expected trophic interactions, this unidentified consumer would have a stable carbon isotope ratio of about 28/mil and a stable nitrogen isotope ratio of about 8/mi. Three possible feeding stages for larval striped bass also were identified, based on their lengths. The smallest length fish seem to subsist on their yolk sac remnants, and the largest length fish subsist on Neomysis shrimp and zooplankton. The intermediate-length fish represent a transition stage between primary food sources and/or use of a mixture of food sources. (USGS)

Longitudinal relaxation of initially straight flexible and stiff polymers

NASA Astrophysics Data System (ADS)

Dimitrakopoulos, Panagiotis; Dissanayake, Inuka

2004-11-01

The present talk considers the relaxation of a single flexible or stiff polymer chain from an initial straight configuration in a viscous solvent. This problem commonly arises when strong flows are turned off in both industrial and biological applications. The problem is also motivated by recent experiments with single biopolymer molecules relaxing after being fully extended by applied forces as well as by the recent development of micro-devices involving stretched tethered biopolymers. Our results are applicable to a wide array of synthetic polymers such as polyacrylamides, Kevlar and polyesters as well as biopolymers such as DNA, actin filaments, microtubules and MTV. In this talk we discuss the mechanism of the polymer relaxation as was revealed through Brownian Dynamics simulations covering a broad range of time scales and chain stiffness. After the short-time free diffusion, the chain's longitudinal reduction at early intermediate times is shown to constitute a universal behavior for any chain stiffness caused by a quasi-steady relaxation of tensions associated with the deforming action of the Brownian forces. Stiff chains are shown to exhibit a late intermediate-time longitudinal reduction associated with a relaxation of tensions affected by the deforming Brownian and the restoring bending forces. The longitudinal and transverse relaxations are shown to obey different laws, i.e. the chain relaxation is anisotropic at all times. In the talk, we show how from the knowledge of the relaxation mechanism, we can predict and explain the polymer properties including the polymer stress and the solution birefringence. In addition, a generalized stress-optic law is derived valid for any time and chain stiffness. All polymer properties which depend on the polymer length are shown to exhibit two intermediate-time behaviors with the early one to constitute a universal behavior for any chain stiffness. This work was supported in part by the Minta Martin Research Fund. The computations were performed on multiprocessor computers provided by the National Center for Supercomputing Applications (NCSA) in Illinois (grant DMR000003), and by an Academic Equipment Grant from Sun Microsystems Inc.

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids[C][W][OPEN

PubMed Central

Niehaus, Thomas D.; Nguyen, Thuy N.D.; Gidda, Satinder K.; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A.; McCarty, Donald R.; Downs, Diana M.; Cooper, Arthur J.L.; Fiehn, Oliver; Mullen, Robert T.; Hanson, Andrew D.

2014-01-01

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase. PMID:25070638

Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease.

PubMed

Zinnanti, William J; Lazovic, Jelena; Griffin, Kathleen; Skvorak, Kristen J; Paul, Harbhajan S; Homanics, Gregg E; Bewley, Maria C; Cheng, Keith C; Lanoue, Kathryn F; Flanagan, John M

2009-04-01

Maple syrup urine disease (MSUD) is an inherited disorder of branched-chain amino acid metabolism presenting with life-threatening cerebral oedema and dysmyelination in affected individuals. Treatment requires life-long dietary restriction and monitoring of branched-chain amino acids to avoid brain injury. Despite careful management, children commonly suffer metabolic decompensation in the context of catabolic stress associated with non-specific illness. The mechanisms underlying this decompensation and brain injury are poorly understood. Using recently developed mouse models of classic and intermediate maple syrup urine disease, we assessed biochemical, behavioural and neuropathological changes that occurred during encephalopathy in these mice. Here, we show that rapid brain leucine accumulation displaces other essential amino acids resulting in neurotransmitter depletion and disruption of normal brain growth and development. A novel approach of administering norleucine to heterozygous mothers of classic maple syrup urine disease pups reduced branched-chain amino acid accumulation in milk as well as blood and brain of these pups to enhance survival. Similarly, norleucine substantially delayed encephalopathy in intermediate maple syrup urine disease mice placed on a high protein diet that mimics the catabolic stress shown to cause encephalopathy in human maple syrup urine disease. Current findings suggest two converging mechanisms of brain injury in maple syrup urine disease including: (i) neurotransmitter deficiencies and growth restriction associated with branched-chain amino acid accumulation and (ii) energy deprivation through Krebs cycle disruption associated with branched-chain ketoacid accumulation. Both classic and intermediate models appear to be useful to study the mechanism of brain injury and potential treatment strategies for maple syrup urine disease. Norleucine should be further tested as a potential treatment to prevent encephalopathy in children with maple syrup urine disease during catabolic stress.

Web Supplement to "Teaching Chain-Weight Real GDP Measures."

ERIC Educational Resources Information Center

Cahill, Miles B.

2003-01-01

Describes a Web site that is a companion to the Miles Cahill article, "Teaching Chain-Weight Real GDP Measures." States that the exercises are useful because intermediate level textbooks treat this topic casually. Indicates that the Web site contains the comment tool, an overview of the concepts, and links to article references. (JEH)

Bending elasticity of macromolecules: analytic predictions from the wormlike chain model.

PubMed

Polley, Anirban; Samuel, Joseph; Sinha, Supurna

2013-01-01

We present a study of the bend angle distribution of semiflexible polymers of short and intermediate lengths within the wormlike chain model. This enables us to calculate the elastic response of a stiff molecule to a bending moment. Our results go beyond the Hookean regime and explore the nonlinear elastic behavior of a single molecule. We present analytical formulas for the bend angle distribution and for the moment-angle relation. Our analytical study is compared against numerical Monte Carlo simulations. The functional forms derived here can be applied to fluorescence microscopic studies on actin and DNA. Our results are relevant to recent studies of "kinks" and cyclization in short and intermediate length DNA strands.

A Motor-Gradient and Clustering Model of the Centripetal Motility of MTOCs in Meiosis I of Mouse Oocytes

PubMed Central

2016-01-01

Asters nucleated by Microtubule (MT) organizing centers (MTOCs) converge on chromosomes during spindle assembly in mouse oocytes undergoing meiosis I. Time-lapse imaging suggests that this centripetal motion is driven by a biased ‘search-and-capture’ mechanism. Here, we develop a model of a random walk in a drift field to test the nature of the bias and the spatio-temporal dynamics of the search process. The model is used to optimize the spatial field of drift in simulations, by comparison to experimental motility statistics. In a second step, this optimized gradient is used to determine the location of immobilized dynein motors and MT polymerization parameters, since these are hypothesized to generate the gradient of forces needed to move MTOCs. We compare these scenarios to self-organized mechanisms by which asters have been hypothesized to find the cell-center- MT pushing at the cell-boundary and clustering motor complexes. By minimizing the error between simulation outputs and experiments, we find a model of “pulling” by a gradient of dynein motors alone can drive the centripetal motility. Interestingly, models of passive MT based “pushing” at the cortex, clustering by cross-linking motors and MT-dynamic instability gradients alone, by themselves do not result in the observed motility. The model predicts the sensitivity of the results to motor density and stall force, but not MTs per aster. A hybrid model combining a chromatin-centered immobilized dynein gradient, diffusible minus-end directed clustering motors and pushing at the cell cortex, is required to comprehensively explain the available data. The model makes experimentally testable predictions of a spatial bias and self-organized mechanisms by which MT asters can find the center of a large cell. PMID:27706163

Interplay between membrane elasticity and active cytoskeleton forces regulates the aggregation dynamics of the immunological synapse

NASA Astrophysics Data System (ADS)

Dharan, Nadiv; Farago, Oded

Adhesion between a T cell and an antigen presenting cell is achieved by TCR-pMHC and LFA1-ICAM1 protein complexes. These segregate to form a special pattern, known as the immunological synapse (IS), consisting of a central quasi-circular domain of TCR-pMHC bonds surrounded by a peripheral domain of LFA1-ICAM1 complexes. Insights gained from imaging studies had led to the conclusion that the formation of the central adhesion domain in the IS is driven by active (ATP-driven) mechanisms. Recent studies, however, suggested that passive (thermodynamic) mechanisms may also play an important role in this process. Here, we present a simple physical model, taking into account the membrane-mediated thermodynamic attraction between the TCR-pMHC bonds and the effective forces that they experience due to ATP-driven actin retrograde flow and transport by dynein motor proteins. Monte Carlo simulations of the model exhibit a good spatio-temporal agreement with the experimentally observed pattern evolution of the TCR-pMHC microclusters. The agreement is lost when one of the aggregation mechanisms is "muted", which helps to identify the respective roles in the process. We conclude that actin retrograde flow drives the centripetal motion of TCR-pMHC bonds, while the membrane-mediated interactions facilitate microcluster formation and growth. In the absence of dynein motors, the system evolves into a ring-shaped pattern, which highlights the role of dynein motors in the formation of the final concentric pattern. The interplay between the passive and active mechanisms regulates the rate of the accumulation process, which in the absence of one them proceeds either too quickly or slowly.

Flagellar oscillation: a commentary on proposed mechanisms.

PubMed

Woolley, David M

2010-08-01

Eukaryotic flagella and cilia have a remarkably uniform internal 'engine' known as the '9+2' axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive 'switching' event is assumed to occur; there are several proposals regarding the nature of the 'switch' and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The 'provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force-generating cycles and dynein-to-dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self-triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.

A Motor-Gradient and Clustering Model of the Centripetal Motility of MTOCs in Meiosis I of Mouse Oocytes.

PubMed

Khetan, Neha; Athale, Chaitanya A

2016-10-01

Asters nucleated by Microtubule (MT) organizing centers (MTOCs) converge on chromosomes during spindle assembly in mouse oocytes undergoing meiosis I. Time-lapse imaging suggests that this centripetal motion is driven by a biased 'search-and-capture' mechanism. Here, we develop a model of a random walk in a drift field to test the nature of the bias and the spatio-temporal dynamics of the search process. The model is used to optimize the spatial field of drift in simulations, by comparison to experimental motility statistics. In a second step, this optimized gradient is used to determine the location of immobilized dynein motors and MT polymerization parameters, since these are hypothesized to generate the gradient of forces needed to move MTOCs. We compare these scenarios to self-organized mechanisms by which asters have been hypothesized to find the cell-center- MT pushing at the cell-boundary and clustering motor complexes. By minimizing the error between simulation outputs and experiments, we find a model of "pulling" by a gradient of dynein motors alone can drive the centripetal motility. Interestingly, models of passive MT based "pushing" at the cortex, clustering by cross-linking motors and MT-dynamic instability gradients alone, by themselves do not result in the observed motility. The model predicts the sensitivity of the results to motor density and stall force, but not MTs per aster. A hybrid model combining a chromatin-centered immobilized dynein gradient, diffusible minus-end directed clustering motors and pushing at the cell cortex, is required to comprehensively explain the available data. The model makes experimentally testable predictions of a spatial bias and self-organized mechanisms by which MT asters can find the center of a large cell.

Organic intermediates in the anaerobic biodegradation of coal to methane under laboratory conditions

USGS Publications Warehouse

Orem, William H.; Voytek, Mary A.; Jones, Elizabeth J.; Lerch, Harry E.; Bates, Anne L.; Corum, Margo D.; Warwick, Peter D.; Clark, Arthur C.

2010-01-01

Organic intermediates in coal fluids produced by anaerobic biodegradation of geopolymers in coal play a key role in the production of methane in natural gas reservoirs. Laboratory biodegradation experiments on sub-bituminous coal from Texas, USA, were conducted using bioreactors to examine the organic intermediates relevant to methane production. Production of methane in the bioreactors was linked to acetate accumulation in bioreactor fluid. Long chain fatty acids, alkanes (C19–C36) and various low molecular weight aromatics, including phenols, also accumulated in the bioreactor fluid and appear to be the primary intermediates in the biodegradation pathway from coal-derived geopolymers to acetate and methane.

Thermodynamic perturbation theory for fused sphere hard chain fluids using nonadditive interactions

NASA Astrophysics Data System (ADS)

Abu-Sharkh, Basel F.; Sunaidi, Abdallah; Hamad, Esam Z.

2004-03-01

A model is developed for the equation of state of fused chains based on Wertheim thermodynamic perturbation theory and nonadditive size interactions. The model also assumes that the structure (represented by the radial distribution function) of the fused chain fluid is the same as that of the touching hard sphere chain fluid. The model is completely based on spherical additive and nonadditive size interactions. The model has the advantage of offering good agreement with simulation data while at the same time being independent of fitted parameters. The model is most accurate for short chains, small values of Δ (slightly fused spheres) and at intermediate (liquidlike) densities.

Folding thermodynamics of pseudoknotted chain conformations

PubMed Central

Kopeikin, Zoia; Chen, Shi-Jie

2008-01-01

We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. PMID:16674261

Somatic insulin signaling regulates a germline starvation response in Drosophila egg chambers

PubMed Central

Burn, K. Mahala; Shimada, Yuko; Ayers, Kathleen; Lu, Feiyue; Hudson, Andrew M.; Cooley, Lynn

2014-01-01

Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers. PMID:25481758

REVIEW ARTICLE: Hither and yon: a review of bi-directional microtubule-based transport

NASA Astrophysics Data System (ADS)

Gross, Steven P.

2004-06-01

Active transport is critical for cellular organization and function, and impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is increasingly clear that many cargos are moved by both sets of motors, and frequently reverse course. This review compares this bi-directional transport to the more well studied uni-directional transport. It discusses some bi-directionally moving cargos, and critically evaluates three different physical models for how such transport might occur. It then considers the evidence for the number of active motors per cargo, and how the net or average direction of transport might be controlled. The likelihood of a complex linking the activities of kinesin and dynein is also discussed. The paper concludes by reviewing elements of apparent universality between different bi-directionally moving cargos and by briefly considering possible reasons for the existence of bi-directional transport.

Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts.

PubMed

Hara, Yuki; Merten, Christoph A

2015-06-08

Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion. Copyright © 2015 Elsevier Inc. All rights reserved.

Maternal dazap2 Regulates Germ Granules by Counteracting Dynein in Zebrafish Primordial Germ Cells.

PubMed

Forbes, Meredyth M; Rothhämel, Sophie; Jenny, Andreas; Marlow, Florence L

2015-07-07

Primordial germ cells (PGCs) are the stem cells of the germline. Generally, germline induction occurs via zygotic factors or the inheritance of maternal determinants called germ plasm (GP). GP is packaged into ribonucleoprotein complexes within oocytes and later promotes the germline fate in embryos. Once PGCs are specified by either mechanism, GP components localize to perinuclear granular-like structures. Although components of zebrafish PGC germ granules have been studied, the maternal factors regulating their assembly and contribution to germ cell development are unknown. Here, we show that the scaffold protein Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and GP assembly, and colocalizes with the GP in oocytes and in PGCs. Mutational analysis revealed a requirement for maternal Dazap2 (MDazap2) in germ-granule maintenance. Through molecular epistasis analyses, we show that MDazap2 is epistatic to Tdrd7 and maintains germ granules in the embryonic germline by counteracting Dynein activity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing

PubMed Central

Hsu, Hsiang-Ting; Viswanath, Dixita I.; Önfelt, Björn

2016-01-01

Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells. PMID:27903610

Mechanism and regulation of rapid telomere prophase movements in mouse meiotic chromosomes

PubMed Central

Lee, Chih-Ying; Horn, Henning F.; Stewart, Colin L.; Burke, Brian; Bolcun-Filas, Ewelina; Schimenti, John C.; Dresser, Michael E.; Pezza, Roberto J.

2015-01-01

SUMMARY Telomere-led rapid prophase movements (RPMs) in meiotic prophase have been observed in diverse eukaryote species. A shared feature of RPMs is that the force that drives the chromosomal movements is transmitted from the cytoskeleton, through the nuclear envelope, to the telomeres. Studies in mice suggested that dynein movement along microtubules is transmitted to telomeres through SUN1/KASH5 nuclear envelope bridges to generate RPMs. We monitored RPMs in mouse seminiferous tubules using four-dimensional fluorescence imaging and quantitative motion analysis to characterize patterns of movement in the RPM process. We find that RPMs reflect a combination of nuclear rotation and individual chromosome movements. The telomeres move along microtubule tracks which are apparently continuous with the cytoskeletal network, and exhibit characteristic arrangements at different stages of prophase. Quantitative measurements confirmed that SUN1/KASH5, microtubules, and dynein but not actin were necessary for RPMs and that defects in meiotic recombination and synapsis resulted in altered RPMs. PMID:25892231

Molecular Motors and Efficient Motion in a Viscoelastic Environment

NASA Astrophysics Data System (ADS)

Bonin, Keith

2005-11-01

Molecular motors perform many critical functions for cells, including chromosome separation during mitosis, vesicle transport, and muscle contraction. In this talk, we will discuss the ways in which physics concepts and instrumentation are being used to determine the forces and efficiencies of two of these motors, kinesin and dynein, in cells. We will emphasize a) studies at Wake Forest University that focus on the force versus velocity curves (load curves) of kinesin in the neurites of live PC12 cells, and b) work at UNC-Chapel Hill that measures the forces developed by dynein motors within beating cilia on the outer surfaces of live lung cells during mucus transport. We will show how the viscoelastic properties of cytoplasm and mucus can be determined from the Brownian motion of vesicles and beads in these media.. We find that the load on these motors in vivo may exceed that in vitro by a factor of 1000, and that several motors can share the task of moving a single vesicle.

Features of the Phosphatidylinositol Cycle and its Role in Signal Transduction.

PubMed

Epand, Richard M

2017-08-01

The phosphatidylinositol cycle (PI-cycle) has a central role in cell signaling. It is the major pathway for the synthesis of phosphatidylinositol and its phosphorylated forms. In addition, some lipid intermediates of the PI-cycle, including diacylglycerol and phosphatidic acid, are also important lipid signaling agents. The PI-cycle has some features that are important for the understanding of its role in the cell. As a cycle, the intermediates will be regenerated. The PI-cycle requires a large amount of metabolic energy. There are different steps of the cycle that occur in two different membranes, the plasma membrane and the endoplasmic reticulum. In order to complete the PI-cycle lipid must be transferred between the two membranes. The role of the Nir proteins in the process has recently been elucidated. The lipid intermediates of the PI-cycle are normally highly enriched with 1-stearoyl-2-arachidonoyl molecular species in mammals. This enrichment will be retained as long as the intermediates are segregated from other lipids of the cell. However, there is a significant fraction (>15 %) of lipids in the PI-cycle of normal cells that have other acyl chains. Phosphatidylinositol largely devoid of arachidonoyl chains are found in cancer cells. Phosphatidylinositol species with less unsaturation will not be as readily converted to phosphatidylinositol-3,4,5-trisphosphate, the lipid required for the activation of Akt with resulting effects on cell proliferation. Thus, the cyclical nature of the PI-cycle, its dependence on acyl chain composition and its requirement for lipid transfer between two membranes, explain many of the biological properties of this cycle.

Paired Helical Filaments from Alzheimer Disease Brain Induce Intracellular Accumulation of Tau Protein in Aggresomes*

PubMed Central

Santa-Maria, Ismael; Varghese, Merina; Ksiȩżak-Reding, Hanna; Dzhun, Anastasiya; Wang, Jun; Pasinetti, Giulio M.

2012-01-01

Abnormal folding of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles, a key neuropathological feature in Alzheimer disease and tauopathies. A specific anatomical pattern of pathological changes developing in the brain suggests that once tau pathology is initiated it propagates between neighboring neuronal cells, possibly spreading along the axonal network. We studied whether PHFs released from degenerating neurons could be taken up by surrounding cells and promote spreading of tau pathology. Neuronal and non-neuronal cells overexpressing green fluorescent protein-tagged tau (GFP-Tau) were treated with isolated fractions of human Alzheimer disease-derived PHFs for 24 h. We found that cells internalized PHFs through an endocytic mechanism and developed intracellular GFP-Tau aggregates with attributes of aggresomes. This was particularly evident by the perinuclear localization of aggregates and redistribution of the vimentin intermediate filament network and retrograde motor protein dynein. Furthermore, the content of Sarkosyl-insoluble tau, a measure of abnormal tau aggregation, increased 3-fold in PHF-treated cells. An exosome-related mechanism did not appear to be involved in the release of GFP-Tau from untreated cells. The evidence that cells can internalize PHFs, leading to formation of aggresome-like bodies, opens new therapeutic avenues to prevent propagation and spreading of tau pathology. PMID:22496370

Investigating the Slow Axonal Transport of Neurofilaments: A Precursor for Optimal Neuronal Signaling

NASA Astrophysics Data System (ADS)

Johnson, Christopher M.

Neurofilaments are the intermediate filaments of neurons and are the most abundant structure of the neuronal cytoskeleton. Once synthesized within the cell body they are then transported throughout the axon along microtubule tracks, driven by the molecular motors kinesin and dynein. This movement is characterized by long pauses with no movement interrupted by infrequent bouts of rapid movement, resulting in an aggregate dense cytoskeletal structure, which serves to regulate an axon's shape and size. Curiously, the modulated kinetics of these polymers produces a very regular, yet non-uniform, morphology in myelinated axons which are composed of discretely spaced myelin-ensheathed segments that are separated by short constricted regions called "nodes of Ranvier". This unique design optimizes the conduction velocity of myelinated axons at minimal fiber size. Hence, neurofilaments regulate the axon caliber to optimize neuron function. The goal of this dissertation is to investigate the motile mechanism of neurofilament transport as well as the resulting electrophysiological effects that follow. We start by examining highly time-resolved kymograph images generated from recorded neurofilament movement via epifluorescence microscopy. Using kymograph analysis, edge detection algorithms, and pixel smoothing tactics, neurofilament trajectories are extracted and used to obtain statistical distributions for the characteristics of how these filaments move within cells. The results suggest that the observed intermittent and bidirectional motions of these filaments might be explained by a model in which dynein and kinesin motors attach to a single neurofilament cargo and interact through mechanical forces only (i.e. a "tug-of-war" model). We test this hypothesis by developing two discrete-state stochastic models for the kinetic cycles of kinesin and dynein, which are then incorporated into a separate stochastic model that represents the posed tug-of-war scenario. We then systematically vary the number of motors in the model and attempt to identify those combinations of motors that show an agreement with the motility characteristic found from the above mentioned kymographs. By pruning the modeled data in accordance with the experimental results, our model can render an estimate of how many motors are attached to the cargo during transport. The model predicts that, on average, the total number of active motors on each neurofilament is relatively small and relatively independent of polymer length, which suggests that the motors may not be distributed uniformly along the filaments. Finally, we develop a model to explore the physiological function of axon morphology sculpted by neurofilament kinetics. Specifically, nodal constrictions are generated by slowing of neurofilaments in the internodal domain (Monsma et al., 2014), but the physiological function of these constrictions is unknown. To address this, we develop a computational model to investigate the effect of nodal constrictions on the axonal conduction velocity. For a fixed number of ion channels, we find that there is an optimal extent of nodal constriction which minimizes the internodal axon caliber that is required to achieve a given target conduction velocity, and we show that this is sensitive to the precise geometry of the axon and myelin sheath in the flanking paranodal regions. Thus axonal constrictions appear to be a biological adaptation that serves to minimize axonal volume, thereby maximizing the spatial and metabolic efficiency of these processes.

Expression, Localization of SUMO-1, and Analyses of Potential SUMOylated Proteins in Bubalus bubalis Spermatozoa

PubMed Central

Brohi, Rahim Dad; Wang, Li; Hassine, Najla Ben; Cao, Jing; Talpur, Hira Sajjad; Wu, Di; Huang, Chun-Jie; Rehman, Zia-Ur; Bhattarai, Dinesh; Huo, Li-Jun

2017-01-01

Mature spermatozoa have highly condensed DNA that is essentially silent both transcriptionally and translationally. Therefore, post translational modifications are very important for regulating sperm motility, morphology, and for male fertility in general. Protein sumoylation was recently demonstrated in human and rodent spermatozoa, with potential consequences for sperm motility and DNA integrity. We examined the expression and localization of small ubiquitin-related modifier-1 (SUMO-1) in the sperm of water buffalo (Bubalus bubalis) using immunofluorescence analysis. We confirmed the expression of SUMO-1 in the acrosome. We further found that SUMO-1 was lost if the acrosome reaction was induced by calcium ionophore A23187. Proteins modified or conjugated by SUMO-1 in water buffalo sperm were pulled down and analyzed by mass spectrometry. Sixty proteins were identified, including proteins important for sperm morphology and motility, such as relaxin receptors and cytoskeletal proteins, including tubulin chains, actins, and dyneins. Forty-six proteins were predicted as potential sumoylation targets. The expression of SUMO-1 in the acrosome region of water buffalo sperm and the identification of potentially SUMOylated proteins important for sperm function implicates sumoylation as a crucial PTM related to sperm function. PMID:28659810

What Have Metabolomics Approaches Taught Us About Type 2 Diabetes?

PubMed

Gonzalez-Franquesa, Alba; Burkart, Alison M; Isganaitis, Elvira; Patti, Mary-Elizabeth

2016-08-01

Type 2 diabetes (T2D) is increasing worldwide, making identification of biomarkers for detection, staging, and effective prevention strategies an especially critical scientific and medical goal. Fortunately, advances in metabolomics techniques, together with improvements in bioinformatics and mathematical modeling approaches, have provided the scientific community with new tools to describe the T2D metabolome. The metabolomics signatures associated with T2D and obesity include increased levels of lactate, glycolytic intermediates, branched-chain and aromatic amino acids, and long-chain fatty acids. Conversely, tricarboxylic acid cycle intermediates, betaine, and other metabolites decrease. Future studies will be required to fully integrate these and other findings into our understanding of diabetes pathophysiology and to identify biomarkers of disease risk, stage, and responsiveness to specific treatments.

Screening and identification of per- and polyfluoroalkyl substances in microwave popcorn bags.

PubMed

Zabaleta, Itsaso; Negreira, Noelia; Bizkarguenaga, Ekhine; Prieto, Ailette; Covaci, Adrian; Zuloaga, Olatz

2017-09-01

Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS) was used for the accurate identification (<10ppm) of different polyfluoroalkylphosphates (PAPs) and their intermediate and end degradation products in popcorn bags. Up to 46 per- and polyfluoroalkyl substances (PFASs) and precursors were identified. Moreover, an accurate method based on focused ultrasonic solid-liquid extraction (FUSLE) and a clean-up step with Envi-Carb sorbent was validated and applied to the quantification of 24 PFASs in popcorn bags from over twelve European countries, three American countries and two Asian countries. To the best of our knowledge, this is the first time that identification and quantification of some intermediates of PFAS precursors (different chain length fluorotelomer saturated acids (FTCAs) and fluorotelomer unsaturated acids (FTUCAs)) have been reported. Moreover, different patterns in the microwave popcorn bag composition were observed within the countries; while in European and American countries short chain PFASs were detected, Asian countries still use long chain PFASs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Impact of amylosucrase modification on the structural and physicochemical properties of native and acid-thinned waxy corn starch.

PubMed

Zhang, Hao; Zhou, Xing; He, Jian; Wang, Tao; Luo, Xiaohu; Wang, Li; Wang, Ren; Chen, Zhengxing

2017-04-01

Recombinant amylosucrase from Neisseria polysaccharea was utilized to modify native and acid-thinned starches. The molecular structures and physicochemical properties of modified starches were investigated. Acid-thinned starch displayed much lower viscosity after gelatinization than did the native starch. However, the enzyme exhibited similar catalytic efficiency for both forms of starch. The modified starches had higher proportions of long (DP>33) and intermediate chains (DP 13-33), and X-ray diffraction showed a B-type crystalline structure for all modified starches. With increasing reaction time, the relative crystallinity and endothermic enthalpy of the modified starches gradually decreased, whereas the melting peak temperatures and resistant starch contents increased. Slight differences were observed in thermal parameters, relative crystallinity, and branch chain length distribution between the modified native and acid-thinned starches. Moreover, the digestibility of the modified starches was not affected by acid hydrolysis pretreatment, but was affected by the percentage of intermediate and long chains. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microbial synthesis of medium-chain chemicals from renewables.

PubMed

Sarria, Stephen; Kruyer, Nicholas S; Peralta-Yahya, Pamela

2017-12-01

Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.

Pentadecanoic and Heptadecanoic Acids: Multifaceted Odd-Chain Fatty Acids12

PubMed Central

Pfeuffer, Maria; Jaudszus, Anke

2016-01-01

The odd-chain fatty acids (OCFAs) pentadecanoic acid (15:0) and heptadecanoic acid (17:0), which account for only a small proportion of total saturated fatty acids in milk fat and ruminant meat, are accepted biomarkers of dairy fat intake. However, they can also be synthesized endogenously, for example, from gut-derived propionic acid (3:0). A number of studies have shown an inverse association between OCFA concentrations in human plasma phospholipids or RBCs and risk of type 2 diabetes and cardiovascular disease. We propose a possible involvement in metabolic regulation from the assumption that there is a link between 15:0 and 17:0 and the metabolism of other short-chain, medium-chain, and longer-chain OCFAs. The OCFAs 15:0 and 17:0 can be elongated to very-long-chain FAs (VLCFAs) such as tricosanoic acid (23:0) and pentacosanoic acid (25:0) in glycosphingolipids, particularly found in brain tissue, or can be derived from these VLCFAs. Their chains can be shortened, yielding propionyl-coenzyme A (CoA). Propionyl-CoA, by succinyl-CoA, can replenish the citric acid cycle (CAC) with anaplerotic intermediates and, thus, improve mitochondrial energy metabolism. Mitochondrial function is compromised in a number of disorders and may be impaired with increasing age. Optimizing anaplerotic intermediate availability for the CAC may help to cope with demands in times of increased metabolic stress and with aging. OCFAs may serve as substrates for synthesis of both odd-numbered VLCFAs and propionyl-CoA or store away excess propionic acid. PMID:27422507

Determination of protein folding kinetic types using sequence and predicted secondary structure and solvent accessibility.

PubMed

Zhang, Hua; Zhang, Tuo; Gao, Jianzhao; Ruan, Jishou; Shen, Shiyi; Kurgan, Lukasz

2012-01-01

Proteins fold through a two-state (TS), with no visible intermediates, or a multi-state (MS), via at least one intermediate, process. We analyze sequence-derived factors that determine folding types by introducing a novel sequence-based folding type predictor called FOKIT. This method implements a logistic regression model with six input features which hybridize information concerning amino acid composition and predicted secondary structure and solvent accessibility. FOKIT provides predictions with average Matthews correlation coefficient (MCC) between 0.58 and 0.91 measured using out-of-sample tests on four benchmark datasets. These results are shown to be competitive or better than results of four modern predictors. We also show that FOKIT outperforms these methods when predicting chains that share low similarity with the chains used to build the model, which is an important advantage given the limited number of annotated chains. We demonstrate that inclusion of solvent accessibility helps in discrimination of the folding kinetic types and that three of the features constitute statistically significant markers that differentiate TS and MS folders. We found that the increased content of exposed Trp and buried Leu are indicative of the MS folding, which implies that the exposure/burial of certain hydrophobic residues may play important role in the formation of the folding intermediates. Our conclusions are supported by two case studies.

Magnetization curves of di-, tri- and tetramerized mixed spin-1 and spin-2 Heisenberg chains

NASA Astrophysics Data System (ADS)

Karľová, Katarína; Strečka, Jozef

2018-05-01

Magnetization curves of ferrimagnetic mixed spin-1 and spin-2 Heisenberg chains are calculated with the help of density-matrix renormalization group method and quantum Monte Carlo simulations by considering a spin dimerization (1,2), trimerization (1,1,2) and tetramerization (1,1,1,2). The investigated mixed-spin Heisenberg chains can be alternatively viewed as a pure spin-1 Heisenberg chain, which contains at a regular lattice positions spin-2 particles. Unlike the antiferromagnetic spin-1 Heisenberg chain solely displaying a zero magnetization plateau due to the Haldane phase, the ferrimagnetic mixed spin-(1,2), spin-(1,1,2) and spin-(1,1,1,2) Heisenberg chains exhibit more striking magnetization curves involving at least two intermediate magnetization plateaux and quantum spin-liquid states.

The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle

PubMed Central

1994-01-01

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein. PMID:8138567

Paramagnetic colloids: Chaotic routes to clusters and molecules

NASA Astrophysics Data System (ADS)

Abdi, Hamed; Soheilian, Rasam; Erb, Randall M.; Maloney, Craig E.

2018-03-01

We present computer simulations and experiments on dilute suspensions of superparamagnetic particles subject to rotating magnetic fields. We focus on chains of four particles and their decay routes to stable structures. At low rates, the chains track the external field. At intermediate rates, the chains break up but perform a periodic (albeit complex) motion. At sufficiently high rates, the chains generally undergo chaotic motion at short times and decay to either closely packed clusters or more dispersed, colloidal molecules at long times. We show that the transition out of the chaotic states can be described as a Poisson process in both simulation and experiment.

Global stocks of selected mineral-based commodities

USGS Publications Warehouse

Wilburn, David R.; Bleiwas, Donald I.; Karl, Nick A.

2016-12-05

IntroductionThe U.S. Geological Survey, National Minerals Information Center, analyzes mineral and metal supply chains by identifying and describing major components of mineral and material flows from ore extraction, through intermediate forms, to a final product. This report focuses on an important component of the world’s supply chain: the amounts and global distribution of major consumer, producer, and exchange stocks of selected mineral commodities. In this report, the term “stock” is used instead of “inventory” and refers to accumulations of mined ore, intermediate products, and refined mineral-based commodities that are in a form that meets the agreed-upon specifications of a buyer or processor of intermediate products. These may include certain ores such as bauxite, concentrates, smelter products, and refined metals. Materials sometimes referred to as inventory for accounting purposes, such as ore contained in a deposit or in a leach pile, or materials that need to be further processed before they can be shipped to a consumer, are not considered. Stocks may be held (owned) by consumers, governments, investors, producers, and traders. They may serve as (1) a means to achieve economic, social, and strategic goals through government policies; (2) a secure source of supply to meet demand and to mitigate potential shortages in the supply chain; (3) a hedge to mitigate price volatility; and (4) vehicles for speculative investment.The paucity and uneven reliability of data for stocks of ores and concentrates and for material held by producers, consumers, and merchants hinder the accurate estimating of the size and distribution of this portion of the supply chain for certain commodities. This paper reviews the more visible stocks held in commodity exchange warehouses distributed throughout the world.

A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

PubMed Central

Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

2016-01-01

To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

A role for the Rab6A′ GTPase in the inactivation of the Mad2-spindle checkpoint

PubMed Central

Miserey-Lenkei, Stéphanie; Couëdel-Courteille, Anne; Del Nery, Elaine; Bardin, Sabine; Piel, Matthieu; Racine, Victor; Sibarita, Jean-Baptiste; Perez, Franck; Bornens, Michel; Goud, Bruno

2006-01-01

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A′, regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A′ function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150Glued, a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A′ likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A′, through its interaction with p150Glued and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition. PMID:16395330

Differential regulation of polarized synaptic vesicle trafficking and synapse stability in neural circuit rewiring in Caenorhabditis elegans

PubMed Central

Kurup, Naina; Kono, Karina

2017-01-01

Neural circuits are dynamic, with activity-dependent changes in synapse density and connectivity peaking during different phases of animal development. In C. elegans, young larvae form mature motor circuits through a dramatic switch in GABAergic neuron connectivity, by concomitant elimination of existing synapses and formation of new synapses that are maintained throughout adulthood. We have previously shown that an increase in microtubule dynamics during motor circuit rewiring facilitates new synapse formation. Here, we further investigate cellular control of circuit rewiring through the analysis of mutants obtained in a forward genetic screen. Using live imaging, we characterize novel mutations that alter cargo binding in the dynein motor complex and enhance anterograde synaptic vesicle movement during remodeling, providing in vivo evidence for the tug-of-war between kinesin and dynein in fast axonal transport. We also find that a casein kinase homolog, TTBK-3, inhibits stabilization of nascent synapses in their new locations, a previously unexplored facet of structural plasticity of synapses. Our study delineates temporally distinct signaling pathways that are required for effective neural circuit refinement. PMID:28636662

LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes.

PubMed

Fu, Meng-Meng; Nirschl, Jeffrey J; Holzbaur, Erika L F

2014-06-09

Autophagy is essential for maintaining cellular homeostasis in neurons, where autophagosomes undergo robust unidirectional retrograde transport along axons. We find that the motor scaffolding protein JIP1 binds directly to the autophagosome adaptor LC3 via a conserved LIR motif. This interaction is required for the initial exit of autophagosomes from the distal axon, for sustained retrograde transport along the midaxon, and for autophagosomal maturation in the proximal axon. JIP1 binds directly to the dynein activator dynactin but also binds to and activates kinesin-1 in a phosphorylation-dependent manner. Following JIP1 depletion, phosphodeficient JIP1-S421A rescues retrograde transport, while phosphomimetic JIP1-S421D aberrantly activates anterograde transport. During normal autophagosome transport, residue S421 of JIP1 may be maintained in a dephosphorylated state by autophagosome-associated MKP1 phosphatase. Moreover, binding of LC3 to JIP1 competitively disrupts JIP1-mediated activation of kinesin. Thus, dual mechanisms prevent aberrant activation of kinesin to ensure robust retrograde transport of autophagosomes along the axon. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of Tryptophan Side Chain Dynamics on the Trp-Cage Mini-Protein Folding Studied by Molecular Dynamics Simulations

PubMed Central

Kannan, Srinivasaraghavan; Zacharias, Martin

2014-01-01

The 20 residue Trp-cage mini-protein is one of smallest proteins that adopt a stable folded structure containing also well-defined secondary structure elements. The hydrophobic core is arranged around a single central Trp residue. Despite several experimental and simulation studies the detailed folding mechanism of the Trp-cage protein is still not completely understood. Starting from fully extended as well as from partially folded Trp-cage structures a series of molecular dynamics simulations in explicit solvent and using four different force fields was performed. All simulations resulted in rapid collapse of the protein to on average relatively compact states. The simulations indicate a significant dependence of the speed of folding to near-native states on the side chain rotamer state of the central Trp residue. Whereas the majority of intermediate start structures with the central Trp side chain in a near-native rotameric state folded successfully within less than 100 ns only a fraction of start structures reached near-native folded states with an initially non-native Trp side chain rotamer state. Weak restraining of the Trp side chain dihedral angles to the state in the folded protein resulted in significant acceleration of the folding both starting from fully extended or intermediate conformations. The results indicate that the side chain conformation of the central Trp residue can create a significant barrier for controlling transitions to a near native folded structure. Similar mechanisms might be of importance for the folding of other protein structures. PMID:24563686

Spot-shadowing optimization to mitigate damage growth in a high-energy-laser amplifier chain.

PubMed

Bahk, Seung-Whan; Zuegel, Jonathan D; Fienup, James R; Widmayer, C Clay; Heebner, John

2008-12-10

A spot-shadowing technique to mitigate damage growth in a high-energy laser is studied. Its goal is to minimize the energy loss and undesirable hot spots in intermediate planes of the laser. A nonlinear optimization algorithm solves for the complex fields required to mitigate damage growth in the National Ignition Facility amplifier chain. The method is generally applicable to any large fusion laser.

Molecular dynamics study of intermediate phase of long chain alkyl sulfonate/water systems.

PubMed

Poghosyan, Armen H; Arsenyan, Levon H; Shahinyan, Aram A

2013-01-08

Using atomic level simulation we aimed to investigate various intermediate phases of the long chain alkyl sulfonate/water system. Overall, about 800 ns parallel molecular dynamics simulation study was conducted for a surfactant/water system consisting of 128 sodium pentadecyl sulfonate and 2251 water molecules. The GROMACS software code with united atom force field was applied. Despite some differences, the analysis of main structural parameters is in agreement with X-ray experimental findings. The mechanism of self-assembly of SPDS molecules was also examined. At T = 323 K we obtained both tilted fully interdigitated and liquid crystalline-like disordered hydrocarbon chains; hence, the presence of either gel phase that coexists with a lamellar phase or metastable gel phase with fraction of gauche configuration can be assumed. Further increase of temperature revealed that the system underwent a transition to a lamellar phase, which was clearly identified by the presence of fully disordered hydrocarbon chains. The transition from gel-to-fluid phase was implemented by simulated annealing treatment, and the phase transition point at T = 335 K was identified. The surfactant force field in its presented set is surely enabled to fully demonstrate the mechanism of self-assembly and the behavior of phase transition making it possible to get important information around the phase transition point.

Modular and selective biosynthesis of gasoline-range alkanes.

PubMed

Sheppard, Micah J; Kunjapur, Aditya M; Prather, Kristala L J

2016-01-01

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Defining Genome Project Standards in a New Era of Sequencing

ScienceCinema

Chain, Patrick

2018-01-16

Patrick Chain of the DOE Joint Genome Institute gives a talk on behalf of the International Genome Sequencing Standards Consortium on the need for intermediate genome classifications between "draft" and "finished".

Natural product biosynthesis: Tackling tunicamycin

NASA Astrophysics Data System (ADS)

Goddard-Borger, Ethan D.; Withers, Stephen G.

2012-07-01

The tunicamycins, secondary metabolites of various Streptomyces species, are invaluable tools in glycobiology. It has now been shown that their biosynthesis involves an unusual exo-glycal intermediate produced by previously unknown short-chain dehydrogenase/reductase activity.

Electron transport chain-dependent and -independent mechanisms of mitochondrial H2O2 emission during long-chain fatty acid oxidation.

PubMed

Seifert, Erin L; Estey, Carmen; Xuan, Jian Y; Harper, Mary-Ellen

2010-02-19

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels.

Total Syntheses of (±)-Ovalicin, C4(S*)-Isomer, and Its C5-Analogs and Anti-trypanosomal Activities∥

PubMed Central

Hua, Duy H.; Zhao, Huiping; Battina, Srinivas K.; Lou, Kaiyan; Jimenez, Ana L.; Desper, John; Perchellet, Elisabeth M.; Perchellet, Jean-Pierre H.; Chiang, Peter K.

2008-01-01

Total syntheses of (±)-ovalicin, its C4(S*)-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(t-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate. Subsequent epoxidation, dihydroxylation, methylation and oxidation led to (3S*,5R*,6R*)-5-methoxy-6-(t-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27) followed by oxidation afforded (±)-ovalicin. The functional group manipulation afforded a number of regio- and stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dictated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evaluated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor cells in vitro. Compounds 46 and a precursor, (3S*,4R*,5R*,6R*)-5-methoxy-4-[(E)-1’,5’-dimethylhexa-1’,4’–dienyl)]-6-(t-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs. PMID:18356059

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, Mark J.; Saleh, Omar A.

We calculated the force-extension curves for a flexible polyelectrolyte chain with varying charge separations by performing Monte Carlo simulations of a 5000 bead chain using a screened Coulomb interaction. At all charge separations, the force-extension curves exhibit a Pincus-like scaling regime at intermediate forces and a logarithmic regime at large forces. As the charge separation increases, the Pincus regime shifts to a larger range of forces and the logarithmic regime starts are larger forces. We also found that force-extension curve for the corresponding neutral chain has a logarithmic regime. Decreasing the diameter of bead in the neutral chain simulations removedmore » the logarithmic regime, and the force-extension curve tends to the freely jointed chain limit. In conclusion, this result shows that only excluded volume is required for the high force logarithmic regime to occur.« less

Intermediates of peroxisomal beta-oxidation. A study of the fatty acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanoate.

PubMed Central

Bartlett, K; Hovik, R; Eaton, S; Watmough, N J; Osmundsen, H

1990-01-01

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation. Images Fig. 2. Fig. 3. Fig. 4. PMID:2396977

An informational transition in conditioned Markov chains: Applied to genetics and evolution.

PubMed

Zhao, Lei; Lascoux, Martin; Waxman, David

2016-08-07

In this work we assume that we have some knowledge about the state of a population at two known times, when the dynamics is governed by a Markov chain such as a Wright-Fisher model. Such knowledge could be obtained, for example, from observations made on ancient and contemporary DNA, or during laboratory experiments involving long term evolution. A natural assumption is that the behaviour of the population, between observations, is related to (or constrained by) what was actually observed. The present work shows that this assumption has limited validity. When the time interval between observations is larger than a characteristic value, which is a property of the population under consideration, there is a range of intermediate times where the behaviour of the population has reduced or no dependence on what was observed and an equilibrium-like distribution applies. Thus, for example, if the frequency of an allele is observed at two different times, then for a large enough time interval between observations, the population has reduced or no dependence on the two observed frequencies for a range of intermediate times. Given observations of a population at two times, we provide a general theoretical analysis of the behaviour of the population at all intermediate times, and determine an expression for the characteristic time interval, beyond which the observations do not constrain the population's behaviour over a range of intermediate times. The findings of this work relate to what can be meaningfully inferred about a population at intermediate times, given knowledge of terminal states. Copyright © 2016 Elsevier Ltd. All rights reserved.

TRAPPC9 mediates the interaction between p150 and COPII vesicles at the target membrane.

PubMed

Zong, Min; Satoh, Ayano; Yu, Mei Kuen; Siu, Ka Yu; Ng, Wing Yan; Chan, Hsiao Chang; Tanner, Julian A; Yu, Sidney

2012-01-01

The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150(Glued), a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown. We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150(Glued) away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150(Glued) via the same carboxyl terminal domain of p150(Glued) that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150(Glued) and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150(Glued) from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane. These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.

A Comparative Proteome Approach Reveals Metabolic Changes Associated with Flammulina velutipes Mycelia in Response to Cold and Light Stress.

PubMed

Liu, Jing-Yu; Chang, Ming-Chang; Meng, Jun-Long; Feng, Cui-Ping; Wang, Yu

2018-04-11

In some industrial processes, cold and light stresses are recognized as two important environmental triggers for the transformation of mycelia into fruit-bodies via intermediate primordia in Flammulina velutipes cultivation. To gain insights into the mechanism of regulation of F. velutipes mycelia in response to cold and light stress, proteins expressed abundantly and characteristically at particular stress states were investigated by using the isobaric tags for the relative and absolute quantitation labeling technique. Among the 1046 nonredundant proteins identified with a high degree of confidence, 264 proteins, which were detected as differentially expressed proteins, were associated with 176 specific KEGG pathways. In-depth data analysis revealed that the regulatory network underlying the cold and light response mechanisms of F. velutipes mycelia was complex and multifaceted, as it included varied functions such as rapid energy supply, the biosynthesis of lysine, phenylalanine, tyrosine, and γ-aminobutyric acid, the calcium signal transduction process, dynein-dependent actin and microtubule cytoskeleton formation, autolysis, oxidative stress adaptation, pigment secretion, tissue and organ morphogenesis, and other interesting stress-related processes. Insights into the proteins might shed light on an intuitive understanding of the cold and light stress response mechanism underlying the fruiting processes of F. velutipes. Furthermore, the data might also provide further insights into the stress response mechanism of macro-fungi and valuable information for scientific improvement of some mushroom cultivation techniques in practice.

Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule

PubMed Central

Rosen, Laura E.; Connell, Katelyn B.; Marqusee, Susan

2014-01-01

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates. PMID:25258414

Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule.

PubMed

Rosen, Laura E; Connell, Katelyn B; Marqusee, Susan

2014-10-14

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.

Backbone amide 15N chemical shift tensors report on hydrogen bonding interactions in proteins: A magic angle spinning NMR study.

PubMed

Paramasivam, Sivakumar; Gronenborn, Angela M; Polenova, Tatyana

2018-08-01

Chemical shift tensors (CSTs) are an exquisite probe of local geometric and electronic structure. 15 N CST are very sensitive to hydrogen bonding, yet they have been reported for very few proteins to date. Here we present experimental results and statistical analysis of backbone amide 15 N CSTs for 100 residues of four proteins, two E. coli thioredoxin reassemblies (1-73-(U- 13 C, 15 N)/74-108-(U- 15 N) and 1-73-(U- 15 N)/74-108-(U- 13 C, 15 N)), dynein light chain 8 LC8, and CAP-Gly domain of the mammalian dynactin. The 15 N CSTs were measured by a symmetry-based CSA recoupling method, ROCSA. Our results show that the principal component δ 11 is very sensitive to the presence of hydrogen bonding interactions due to its unique orientation in the molecular frame. The downfield chemical shift change of backbone amide nitrogen nuclei with increasing hydrogen bond strength is manifested in the negative correlation of the principal components with hydrogen bond distance for both α-helical and β-sheet secondary structure elements. Our findings highlight the potential for the use of 15 N CSTs in protein structure refinement. Copyright © 2018 Elsevier Inc. All rights reserved.

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

PubMed

De Rossi, María Cecilia; Wetzler, Diana E; Benseñor, Lorena; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Gelfand, Vladimir; Bruno, Luciana; Levi, Valeria

2017-12-01

Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells. We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-β-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios. The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams. Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport. Copyright © 2017 Elsevier B.V. All rights reserved.

Different modes of APC/C activation control growth and neuron-glia interaction in the developing Drosophila eye.

PubMed

Neuert, Helen; Yuva-Aydemir, Yeliz; Silies, Marion; Klämbt, Christian

2017-12-15

The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins, Fizzy/Cdc20 and Fizzy-related/Cdh1, confer APC/C substrate specificity. Here, we show that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, we identified a series of novel APC/C candidate substrates. Four of our candidate genes are required for fzr/cdh1 -dependent neuron-glia interaction, including the dynein light chain Dlc90F Taken together, our data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development. © 2017. Published by The Company of Biologists Ltd.

Mutations in DYNC1H1 cause severe intellectual disability with neuronal migration defects.

PubMed

Willemsen, Marjolein H; Vissers, Lisenka E L; Willemsen, Michèl A A P; van Bon, Bregje W M; Kroes, Thessa; de Ligt, Joep; de Vries, Bert B; Schoots, Jeroen; Lugtenberg, Dorien; Hamel, Ben C J; van Bokhoven, Hans; Brunner, Han G; Veltman, Joris A; Kleefstra, Tjitske

2012-03-01

DYNC1H1 encodes the heavy chain protein of the cytoplasmic dynein 1 motor protein complex that plays a key role in retrograde axonal transport in neurons. Furthermore, it interacts with the LIS1 gene of which haploinsufficiency causes a severe neuronal migration disorder in humans, known as classical lissencephaly or Miller-Dieker syndrome. To describe the clinical spectrum and molecular characteristics of DYNC1H1 mutations. A family based exome sequencing approach was used to identify de novo mutations in patients with severe intellectual disability. In this report the identification of two de novo missense mutations in DYNC1H1 (p.Glu1518Lys and p.His3822Pro) in two patients with severe intellectual disability and variable neuronal migration defects is described. Since an autosomal dominant mutation in DYNC1H1 was previously identified in a family with the axonal (type 2) form of Charcot- Marie-Tooth (CMT2) disease and mutations in Dync1h1 in mice also cause impaired neuronal migration in addition to neuropathy, these data together suggest that mutations in DYNC1H1 can lead to a broad phenotypic spectrum and confirm the importance of DYNC1H1 in both central and peripheral neuronal functions.

Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia.

PubMed

Shi, Wengui; Xie, Yanfang; He, Jinpeng; Zhou, Jian; Gao, Yuhai; Wei, Wenjun; Ding, Nan; Ma, Huiping; Xian, Cory J; Chen, Keming; Wang, Jufang

2017-05-12

It is well documented that microgravity in space environment leads to bone loss in astronauts. These physiological changes have also been validated by human and animal studies and modeled in cell-based analogs. However, the underlying mechanisms are elusive. In the current study, we identified a novel phenomenon that primary cilia (key sensors and functioning organelles) of rat calvarial osteoblasts (ROBs) gradually shrank and disappeared almost completely after exposure to simulated microgravity generated by a random positioning machine (RPM). Along with the abrogation of primary cilia, the differentiation, maturation and mineralization of ROBs were inhibited. We also found that the disappearance of primary cilia was prevented by treating ROBs with cytochalasin D, but not with LiCl or dynein light chain Tctex-type 1 (Dynlt1) siRNA. The repression of the differentiation, maturation and mineralization of ROBs was effectively offset by cytochalasin D treatment in microgravity conditions. Blocking ciliogenesis using intraflagellar transport protein 88 (IFT88) siRNA knockdown inhibited the ability of cytochalasin D to counteract this reduction of osteogenesis. These results indicate that the abrogation of primary cilia may be responsible for the microgravity's inhibition on osteogenesis. Reconstruction of primary cilia may become a potential strategy against bone loss induced by microgravity.

An exploration in mineral supply chain mapping using tantalum as an example

USGS Publications Warehouse

Soto-Viruet, Yadira; Menzie, W. David; Papp, John F.; Yager, Thomas R.

2013-01-01

This report uses the supply chain of tantalum (Ta) to investigate the complexity of mineral and metal supply chains in general and show how they can be mapped. A supply chain is made up of all the manufacturers, suppliers, information networks, and so forth, that provide the materials and parts that go into making up a final product. The mineral portion of the supply chain begins with mineral material in the ground (the ore deposit); extends through a series of processes that include mining, beneficiation, processing (smelting and refining), semimanufacture, and manufacture; and continues through transformation of the mineral ore into concentrates, refined mineral commodities, intermediate forms (such as metals and alloys), component parts, and, finally, complex products. This study analyses the supply chain of tantalum beginning with minerals in the ground to many of the final goods that contain tantalum.

Tandem catalysis for the preparation of cylindrical polypeptide brushes.

PubMed

Rhodes, Allison J; Deming, Timothy J

2012-11-28

Here, we report a method for synthesis of cylindrical copolypeptide brushes via N-carboxyanhydride (NCA) polymerization utilizing a new tandem catalysis approach that allows preparation of brushes with controlled segment lengths in a straightforward, one-pot procedure requiring no intermediate isolation or purification steps. To obtain high-density brush copolypeptides, we used a "grafting from" approach where alloc-α-aminoamide groups were installed onto the side chains of NCAs to serve as masked initiators. These groups were inert during cobalt-initiated NCA polymerization and gave allyloxycarbonyl-α-aminoamide-substituted polypeptide main chains. The alloc-α-aminoamide groups were then activated in situ using nickel to generate initiators for growth of side-chain brush segments. This use of stepwise tandem cobalt and nickel catalysis was found to be an efficient method for preparation of high-chain-density, cylindrical copolypeptide brushes, where both the main chains and side chains can be prepared with controlled segment lengths.

Genetic Diversity of Plasmodium falciparum Merozoite Surface Protein-1 Block 2 in Sites of Contrasting Altitudes and Malaria Endemicities in the Mount Cameroon Region

PubMed Central

Wanji, Samuel; Kengne-Ouafo, Arnaud J.; Joan Eyong, Ebanga E.; Kimbi, Helen K.; Tendongfor, Nicholas; Ndamukong-Nyanga, Judith L.; Nana-Djeunga, Hugues C.; Bourguinat, Catherine; Sofeu-Feugaing, David D.; Charvet, Claude L.

2012-01-01

The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/circumsporozoite protein–enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction–based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms. PMID:22556072

Genetic diversity of Plasmodium falciparum merozoite surface protein-1 block 2 in sites of contrasting altitudes and malaria endemicities in the Mount Cameroon region.

PubMed

Wanji, Samuel; Kengne-Ouafo, Arnaud J; Eyong, Ebanga E Joan; Kimbi, Helen K; Tendongfor, Nicholas; Ndamukong-Nyanga, Judith L; Nana-Djeunga, Hugues C; Bourguinat, Catherine; Sofeu-Feugaing, David D; Charvet, Claude L

2012-05-01

The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/circumsporozoite protein-enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction-based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms.

Analysis of the intermediate size proteoglycans from the developing chick limb buds.

PubMed

Vasan, N

1982-08-01

Limb-bud proteoglycans are heterogeneous molecules which vary in their chemical and physical properties with development. This report describes proteoglycan intermediates (PG-I) that predominate in stage-34 limbs, and compares them with proteoglycan aggregates (PG-A) in stage-38 limbs. We analysed proteoglycans and their components extracted with guanidinium chloride by subjecting them to density gradient centrifugation, molecular sieve chromatography, electrophoretic separation, and selective enzymatic degradation. PG-I and PG-A have similar chondroitin sulphate composition, amino sugars, chondroitin sulphate side-chain length, glycoprotein link factors, and hyaluronic acid binding capacity, and both cross react with antisera prepared against cartilage-specific chick sternal proteoglycans. However, PG-I has lower molecular weight, lower buoyant density, and fewer chondroitin sulphate side chains on the protein core. The PG-I in the developing limb can be considered a mixture of smaller aggregates and cartilage-specific large monomers in which the former predominate.

[Healthcare value chain: a model for the Brazilian healthcare system].

PubMed

Pedroso, Marcelo Caldeira; Malik, Ana Maria

2012-10-01

This article presents a model of the healthcare value chain which consists of a schematic representation of the Brazilian healthcare system. The proposed model is adapted for the Brazilian reality and has the scope and flexibility for use in academic activities and analysis of the healthcare sector in Brazil. It places emphasis on three components: the main activities of the value chain, grouped in vertical and horizontal links; the mission of each link and the main value chain flows. The proposed model consists of six vertical and three horizontal links, amounting to nine. These are: knowledge development; supply of products and technologies; healthcare services; financial intermediation; healthcare financing; healthcare consumption; regulation; distribution of healthcare products; and complementary and support services. Four flows can be used to analyze the value chain: knowledge and innovation; products and services; financial; and information.

Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, J.; Warby, C; Whitby, F

2009-01-01

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connectedmore » by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.« less

Detailed dimethylacetal and fatty acid composition of rumen content from lambs fed lucerne or concentrate supplemented with soybean oil.

PubMed

Alves, Susana P; Santos-Silva, José; Cabrita, Ana R J; Fonseca, António J M; Bessa, Rui J B

2013-01-01

Lipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18:1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18:2n-6 and 18:3n-3. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18:0 might be produced during biohydrogenation of the 18:3n-3.

How molecular motors shape the flagellar beat

PubMed Central

Riedel-Kruse, Ingmar H.; Hilfinger, Andreas; Howard, Jonathon; Jülicher, Frank

2007-01-01

Cilia and eukaryotic flagella are slender cellular appendages whose regular beating propels cells and microorganisms through aqueous media. The beat is an oscillating pattern of propagating bends generated by dynein motor proteins. A key open question is how the activity of the motors is coordinated in space and time. To elucidate the nature of this coordination we inferred the mechanical properties of the motors by analyzing the shape of beating sperm: Steadily beating bull sperm were imaged and their shapes were measured with high precision using a Fourier averaging technique. Comparing our experimental data with wave forms calculated for different scenarios of motor coordination we found that only the scenario of interdoublet sliding regulating motor activity gives rise to satisfactory fits. We propose that the microscopic origin of such “sliding control” is the load dependent detachment rate of motors. Agreement between observed and calculated wave forms was obtained only if significant sliding between microtubules occurred at the base. This suggests a novel mechanism by which changes in basal compliance could reverse the direction of beat propagation. We conclude that the flagellar beat patterns are determined by an interplay of the basal properties of the axoneme and the mechanical feedback of dynein motors. PMID:19404446

Branchial Cilia and Sperm Flagella Recruit Distinct Axonemal Components

PubMed Central

Konno, Alu; Shiba, Kogiku; Cai, Chunhua; Inaba, Kazuo

2015-01-01

Eukaryotic cilia and flagella have highly conserved 9 + 2 structures. They are functionally diverged to play cell-type-specific roles even in a multicellular organism. Although their structural components are therefore believed to be common, few studies have investigated the molecular diversity of the protein components of the cilia and flagella in a single organism. Here we carried out a proteomic analysis and compared protein components between branchial cilia and sperm flagella in a marine invertebrate chordate, Ciona intestinalis. Distinct feature of protein recruitment in branchial cilia and sperm flagella has been clarified; (1) Isoforms of α- and β-tubulins as well as those of actins are distinctly used in branchial cilia or sperm flagella. (2) Structural components, such as dynein docking complex, tektins and an outer dense fiber protein, are used differently by the cilia and flagella. (3) Sperm flagella are specialized for the cAMP- and Ca2+-dependent regulation of outer arm dynein and for energy metabolism by glycolytic enzymes. Our present study clearly demonstrates that flagellar or ciliary proteins are properly recruited according to their function and stability, despite their apparent structural resemblance and conservation. PMID:25962172

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

PubMed

Goshima, Gohta; Nédélec, François; Vale, Ronald D

2005-10-24

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

DISC1, PDE4B, and NDE1 at the centrosome and synapse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradshaw, Nicholas J.; Ogawa, Fumiaki; Antolin-Fontes, Beatriz

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependantmore » Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.« less

Involvement of Dynein and Spectrin with Early Melanosome Transport and Melanosomal Protein Trafficking

PubMed Central

Watabe, Hidenori; Valencia, Julio C.; Le Pape, Elodie; Yamaguchi, Yuji; Nakamura, Masayuki; Rouzaud, François; Hoashi, Toshihiko; Kawa, Yoko; Mizoguchi, Masako; Hearing, Vincent J.

2007-01-01

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, TYR and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system which is involved with the sorting of cargo from the plasma membrane. These results provide important clues towards understanding the processes involved with early events in melanosome formation and transport. PMID:17687388

Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

PubMed Central

Di Giovanni, Jerome; Sheng, Zu-Hang

2015-01-01

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca2+ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca2+ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting. PMID:26108535

A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

PubMed Central

Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

2015-01-01

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. DOI: http://dx.doi.org/10.7554/eLife.05697.001 PMID:25898002

Tug of war of molecular motors: the effects of uneven load sharing

NASA Astrophysics Data System (ADS)

Bouzat, Sebastián; Falo, Fernando

2011-12-01

We analyze theoretically the problem of cargo transport along microtubules by motors of two species with opposite polarities. We consider two different one-dimensional models previously developed in the literature: a quite widespread model which assumes equal force sharing, here referred to as the mean field model (MFM), and a stochastic model (SM) which considers individual motor-cargo links. We find that in generic situations, the MFM predicts larger cargo mean velocity, smaller mean run time and less frequent reversions than the SM. These phenomena are found to be the consequences of the load sharing assumptions and can be interpreted in terms of the probabilities of the different motility states. We also explore the influence of the viscosity in both models and the role of the stiffness of the motor-cargo links within the SM. Our results show that the mean cargo velocity is independent of the stiffness, while the mean run time decreases with such a parameter. We explore the case of symmetric forward and backward motors considering kinesin-1 parameters, and the problem of transport by kinesin-1 and cytoplasmic dyneins considering two different sets of parameters previously proposed for dyneins.

Rab5 and its effector FHF contribute to neuronal polarity through dynein-dependent retrieval of somatodendritic proteins from the axon

PubMed Central

Guo, Xiaoli; Farías, Ginny G.; Mattera, Rafael; Bonifacino, Juan S.

2016-01-01

An open question in cell biology is how the general intracellular transport machinery is adapted to perform specialized functions in polarized cells such as neurons. Here we illustrate this adaptation by elucidating a role for the ubiquitous small GTPase Ras-related protein in brain 5 (Rab5) in neuronal polarity. We show that inactivation or depletion of Rab5 in rat hippocampal neurons abrogates the somatodendritic polarity of the transferrin receptor and several glutamate receptor types, resulting in their appearance in the axon. This loss of polarity is not caused primarily by increased transport from the soma to the axon but rather by decreased retrieval from the axon to the soma. Retrieval is also dependent on the Rab5 effector Fused Toes (FTS)–Hook–FTS and Hook-interacting protein (FHIP) (FHF) complex, which interacts with the minus-end–directed microtubule motor dynein and its activator dynactin to drive a population of axonal retrograde carriers containing somatodendritic proteins toward the soma. These findings emphasize the importance of both biosynthetic sorting and axonal retrieval for the polarized distribution of somatodendritic receptors at steady state. PMID:27559088

Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation*

PubMed Central

Seifert, Erin L.; Estey, Carmen; Xuan, Jian Y.; Harper, Mary-Ellen

2010-01-01

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels. PMID:20032466

End-monomer Dynamics in Semiflexible Polymers

PubMed Central

Hinczewski, Michael; Schlagberger, Xaver; Rubinstein, Michael; Krichevsky, Oleg; Netz, Roland R.

2009-01-01

Spurred by an experimental controversy in the literature, we investigate the end-monomer dynamics of semiflexible polymers through Brownian hydrodynamic simulations and dynamic mean-field theory. Precise experimental observations over the last few years of end-monomer dynamics in the diffusion of double-stranded DNA have given conflicting results: one study indicated an unexpected Rouse-like scaling of the mean squared displacement (MSD) 〈r2(t)〉 ~ t1/2 at intermediate times, corresponding to fluctuations at length scales larger than the persistence length but smaller than the coil size; another study claimed the more conventional Zimm scaling 〈r2(t)〉 ~ t2/3 in the same time range. Using hydrodynamic simulations, analytical and scaling theories, we find a novel intermediate dynamical regime where the effective local exponent of the end-monomer MSD, α(t) = d log〈r2(t)〉/d log t, drops below the Zimm value of 2/3 for sufficiently long chains. The deviation from the Zimm prediction increases with chain length, though it does not reach the Rouse limit of 1/2. The qualitative features of this intermediate regime, found in simulations and in an improved mean-field theory for semiflexible polymers, in particular the variation of α(t) with chain and persistence lengths, can be reproduced through a heuristic scaling argument. Anomalously low values of the effective exponent α are explained by hydrodynamic effects related to the slow crossover from dynamics on length scales smaller than the persistence length to dynamics on larger length scales. PMID:21359118

Short, intermediate and mesoscopic range order in sulfur-rich binary glasses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bychkov, E.; Miloshova, M.; Price, D.L.

2008-09-29

Pulsed neutron and high-energy X-ray diffraction, small-angle neutron scattering, Raman spectroscopy and DSC were used to study structural changes on the short, intermediate and mesoscopic range scale for sulfur-rich AsS{sub x} (x {ge} 1.5) and GeS{sub x} (x {ge} 2) glasses. Two structural regions were found in the both systems. (1) Between stoichiometric (As{sub 2}S{sub 3} and GeS{sub 2}) and 'saturated' (AsS{sub 2.2} and GeS{sub 2.7}) compositions, excessive sulfur atoms form sulfur dimers and/or short chains, replacing bridging sulfur in corner-sharing AsS{sub 3/2} and GeS{sub 4/2} units. (2) Above the 'saturated' compositions at [As] < 30.5 at.% and [Ge]

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage*

PubMed Central

Comeaux, Evan Q.; Cuya, Selma M.; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C.; Mobley, James A.; Bjornsti, Mary-Ann; van Waardenburg, Robert C. A. M.

2015-01-01

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

Long chain fatty acids and related pro-inflammatory, specialized pro-resolving lipid mediators and their intermediates in preterm human milk during the first month of lactation.

PubMed

Robinson, D T; Palac, H L; Baillif, V; Van Goethem, E; Dubourdeau, M; Van Horn, L; Martin, C R

2017-06-01

This study aimed to measure longitudinal quantities of the long chain fatty acids, their biologically active terminal metabolites and related intermediates (also called oxylipins) in preterm human milk expressed during the first month of lactation. In a prospective cohort, breast milk was collected throughout the first month of lactation in 30 women who delivered preterm infants. Eighteen bioactive lipids and their intermediates were quantified via solid phase extraction and LC-MS/MS. Analysis by GC-FID quantified the fatty acid precursors. Arachidonic acid (ARA) and docosahexaenoic acid (DHA) milk concentrations significantly declined throughout the first month. Oxylipin concentrations did not change during lactation. Positive associations existed between ARA and thromboxane B2, eicosapentaenoic acid and 18-hydroxyeicosapentaenoic acid, and between DHA and PDX and 14- and 17-hydroxydocosahexaenoic acids. DHA concentrations were 1.5 times higher and 14-HDHA was 1.7 times higher in milk from women taking DHA supplements. This investigation showed conditionally essential fatty acids, ARA and DHA, decreased in preterm milk, suggesting a need to supplement their intake for the breast milk-fed preterm infant. Positive associations between parent fatty acids, bioactive lipids and intermediates, as well as sensitivity of milk to maternal fatty acid intake, support consideration of a comprehensive approach to providing fatty acids for preterm infants through both maternal and infant supplementation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer.

PubMed

He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L; Mann, Colin J; Augst, Ryan; Morris, Charles D; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B; Burton, Dennis R; Zhu, Jiang

2017-01-01

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer

PubMed Central

He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L.; Mann, Colin J.; Augst, Ryan; Morris, Charles D.; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B.; Burton, Dennis R.; Zhu, Jiang

2017-01-01

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development. PMID:28883821

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

PubMed Central

Surana, Parag

2016-01-01

Abstract The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process. PMID:27111887

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology.

PubMed

Armen, Roger S; Daggett, Valerie

2005-12-13

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.

A Study of the Nephrotoxicity and Metabolism of Tetralin and Indan in Fischer 344 Rats

DTIC Science & Technology

1989-05-01

5, a jet fuel composed of aliphatic and aromatic hydrocarbons with the majority of the straight-chain hydrocarbons being between C10 and C15...hydrocarbon of intermediate boiling point and volatility and is similar to the civilian jet fuel , A-1. DFM is a mixture of long chain aliphatic...to compliment earlier research on jet fuel , JP-10, cis- and trans- decalin, and tetralin, it was anticipated some enlightenment could be obtained on

Simulations of stretching a flexible polyelectrolyte with varying charge separation

DOE PAGES

Stevens, Mark J.; Saleh, Omar A.

2016-07-22

We calculated the force-extension curves for a flexible polyelectrolyte chain with varying charge separations by performing Monte Carlo simulations of a 5000 bead chain using a screened Coulomb interaction. At all charge separations, the force-extension curves exhibit a Pincus-like scaling regime at intermediate forces and a logarithmic regime at large forces. As the charge separation increases, the Pincus regime shifts to a larger range of forces and the logarithmic regime starts are larger forces. We also found that force-extension curve for the corresponding neutral chain has a logarithmic regime. Decreasing the diameter of bead in the neutral chain simulations removedmore » the logarithmic regime, and the force-extension curve tends to the freely jointed chain limit. In conclusion, this result shows that only excluded volume is required for the high force logarithmic regime to occur.« less

Biodegradation of long-chain n-paraffins from waste oil of car engine by Acinetobacter sp.

PubMed

Koma, D; Hasumi, F; Yamamoto, E; Ohta, T; Chung, S Y; Kubo, M

2001-01-01

Microorganisms that degrade long-chain n-paraffins from used car engine oil were isolated from soil. For the screening, a fraction of n-paraffin prepared from car engine oil was applied as the sole carbon source. The strain was identified as Acinetobacter sp. The ability of the strain to assimilate long-chain n-paraffins was assessed and characterized. The strain mineralized long-chain n-paraffins (0.1% w/v) in the minimal medium after cultivation for 96 h and also reduced the weight of the waste oil added (1% w/v) by 20% after 72 h without an extracellular biosurfactant. When n-hexadecane was fed as substrate, 1-hexadecanol and 1-hexadecanoic acid were detected as the intermediates by gas chromatography/mass spectrometry. This indicates that the long-chain n-paraffins were metabolized via the terminal oxidation pathway of n-alkane.

The intermediate wavelength magnetic anomaly field of the north Pacific and possible source distributions

NASA Technical Reports Server (NTRS)

Labrecque, J. L.; Cande, S. C.; Jarrard, R. D. (Principal Investigator)

1983-01-01

A technique that eliminates external field sources and the effects of strike aliasing was used to extract from marine survey data the intermediate wavelength magnetic anomaly field for (B) in the North Pacific. A strong correlation exists between this field and the MAGSAT field although a directional sensitivity in the MAGSAT field can be detected. The intermediate wavelength field is correlated to tectonic features. Island arcs appear as positive anomalies of induced origin likely due to variations in crustal thickness. Seamount chains and oceanic plateaus also are manifested by strong anomalies. The primary contribution to many of these anomalies appears to be due to a remanent magnetization. The source parameters for the remainder of these features are presently unidentified ambiguous. Results indicate that the sea surface field is a valuable source of information for secular variation analysis and the resolution of intermediate wavelength source parameters.

Crystallization in diblock copolymer thin films at different degrees of supercooling

NASA Astrophysics Data System (ADS)

Darko, C.; Botiz, I.; Reiter, G.; Breiby, D. W.; Andreasen, J. W.; Roth, S. V.; Smilgies, D.-M.; Metwalli, E.; Papadakis, C. M.

2009-04-01

The crystalline structures in thin films of polystyrene- b -poly(ethylene oxide) (PS- b -PEO) diblock copolymers were studied in dependence on the degree of supercooling. Atomic force microscopy showed that the crystalline domains (lamellae) consist of grains, which are macroscopic at low and intermediate degrees of supercooling, but of submicrometer size for strong supercooling. Using grazing-incidence wide-angle x-ray scattering, we could determine the grain orientation distribution function which shows that the chain stems are perpendicular to the lamellae at low supercooling, but tilted at intermediate and strong supercooling. These results suggest that, at intermediate and strong supercooling, the crystalline PEO lamellae do not grow homogeneously, but by the formation of small crystallites at the growth front.

J chain and myocyte enhancer factor 2B are useful in differentiating classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma and primary mediastinal large B-cell lymphoma.

PubMed

Moore, Erika M; Swerdlow, Steven H; Gibson, Sarah E

2017-10-01

Although most classical Hodgkin lymphomas (CHLs) are easily distinguished from nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and primary mediastinal large B-cell lymphoma (PMBL), cases with significant CD20 expression cause diagnostic confusion. Although the absence of OCT-2 and BOB.1 are useful in these circumstances, a variable proportion of CHLs are positive for these antigens. We investigated the utility of J chain and myocyte enhancer factor 2B (MEF2B) in the diagnosis of CHL; NLPHL; PMBL; T-cell/histiocyte-rich large B-cell lymphoma (TCRLBL); and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, compared with OCT-2 and BOB.1. J chain and MEF2B highlighted lymphocyte predominant (LP) cells in 20/20 (100%) NLPHLs and were negative in 43/43 (100%) CHLs. Fourteen of 15 (93%) PMBLs and 4/4 (100%) TCRLBLs were MEF2B positive, whereas 67% of PMBLs and 50% of TCRLBLs were J chain positive. Three of 3 B-cell lymphomas, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, were negative for J chain and MEF2B. J chain and MEF2B were 100% sensitive and specific for NLPHL versus CHL. MEF2B was 100% sensitive and 98% specific for PMBL versus CHL. Whereas loss of OCT-2 and/or BOB.1 expression had a sensitivity of only 86% and specificity of 100% for CHL versus NLPHL, PMBL, and TCRLBL, lack of both J chain and MEF2B expression was 100% sensitive and 97% specific. J chain and MEF2B are highly sensitive and specific markers of NLPHL versus CHL; are particularly useful in highlighting LP cells; and, with rare exception, are of greater utility than OCT-2 and BOB.1 in differentiating CHL from NLPHL and other large B-cell lymphomas. Copyright © 2017 Elsevier Inc. All rights reserved.

The folding pathways and thermodynamics of semiflexible polymers

NASA Astrophysics Data System (ADS)

Wu, Jing; Cheng, Chenqian; Liu, Gaoyuan; Zhang, Ping; Chen, Tao

2018-05-01

Inspired by the protein folding and DNA packing, we have systematically studied the thermodynamic and kinetic behaviors of single semiflexible homopolymers by Langevin dynamics simulations. In line with experiments, a rich variety of folding products, such as rod-like bundles, hairpins, toroids, and a mixture of them, are observed in the complete diagram of states. Moreover, knotted structures with a significant population are found in a certain range of bending stiffness in thermal equilibrium. As the solvent quality becomes poorer, the population of the intermediate occurring in the folding process increases, which leads to a severe chevron rollover for the folding arm. However, the population of the intermediates in the unfolding process is very low, insufficient to induce unfolding arm rollover. The total types of folding pathways from the coil state to the toroidal state for a semiflexible polymer chain remain unchanged by varying the solvent quality or temperature, whereas the kinetic partitioning into different folding events can be tuned significantly. In the process of knotting, three types of mechanisms, namely, plugging, slipknotting, and sliding, are discovered. Along the folding evolution, a semiflexible homopolymer chain can knot at any stage of folding upon leaving the extended coil state, and the probability to find a knot increases with chain compactness. In addition, we find rich types of knotted topologies during the folding of a semiflexible homopolymer chain. This study should be helpful in gaining insight into the general principles of biopolymer folding.

Detailed Dimethylacetal and Fatty Acid Composition of Rumen Content from Lambs Fed Lucerne or Concentrate Supplemented with Soybean Oil

PubMed Central

Alves, Susana P.; Santos-Silva, José; Cabrita, Ana R. J.; Fonseca, António J. M.; Bessa, Rui J. B.

2013-01-01

Lipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18∶1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18∶2n−6 and 18∶3n−3. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18∶0 might be produced during biohydrogenation of the 18∶3n−3. PMID:23484024

Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.

PubMed

Dridi, Kaouthar; Amara, Sawsan; Bezzine, Sofiane; Rodriguez, Jorge A; Carrière, Frédéric; Gaussier, Hélène

2013-07-01

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.

Top predators induce the evolutionary diversification of intermediate predator species.

PubMed

Zu, Jian; Yuan, Bo; Du, Jianqiang

2015-12-21

We analyze the evolutionary branching phenomenon of intermediate predator species in a tritrophic food chain model by using adaptive dynamics theory. Specifically, we consider the adaptive diversification of an intermediate predator species that feeds on a prey species and is fed upon by a top predator species. We assume that the intermediate predator׳s ability to forage on the prey can adaptively improve, but this comes at the cost of decreased defense ability against the top predator. First, we identify the general properties of trade-off relationships that lead to a continuously stable strategy or to evolutionary branching in the intermediate predator species. We find that if there is an accelerating cost near the singular strategy, then that strategy is continuously stable. In contrast, if there is a mildly decelerating cost near the singular strategy, then that strategy may be an evolutionary branching point. Second, we find that after branching has occurred, depending on the specific shape and strength of the trade-off relationship, the intermediate predator species may reach an evolutionarily stable dimorphism or one of the two resultant predator lineages goes extinct. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

PubMed

Robinson, Philip J; Pringle, Marie Anne; Woolhead, Cheryl A; Bulleid, Neil J

2017-04-28

The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chain elongation and cyclization in type III PKS DpgA.

PubMed

Wu, Hai-Chen; Li, Yi-San; Liu, Yu-Chen; Lyu, Syue-Yi; Wu, Chang-Jer; Li, Tsung-Lin

2012-04-16

Chain elongation and cyclization of precursors of dihydroxyphenylacetyl-CoA (DPA-CoA) catalyzed by the bacterial type III polyketide synthase DpgA were studied. Two labile intermediates, di- and tri-ketidyl-CoA (DK- and TK-CoA), were proposed and chemically synthesized. In the presence of DpgABD, each of these with [(13)C(3)]malonyl-CoA (MA-CoA) was able to form partially (13)C-enriched DPA-CoA. By NMR and MS analysis, the distribution of (13)C atoms in the partially (13)C-enriched DPA-CoA shed light on how the polyketide chain elongates and cyclizes in the DpgA-catalyzed reaction. Polyketone intermediates elongate in a manner different from that which had been believed: two molecules of DK-CoA, or one DK-CoA plus one acetoacetyl-CoA (AA-CoA), but not two molecules of AA-CoA can form one molecule of DPA-CoA. As a result, polyketidyl-CoA serves as both the starter and extender, whereas polyketone-CoA without the terminal carboxyl group can only act as an extender. The terminal carboxyl group is crucial for the cyclization that likely takes place on CoA. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Climate change enhances the negative effects of predation risk on an intermediate consumer.

PubMed

Miller, Luke P; Matassa, Catherine M; Trussell, Geoffrey C

2014-12-01

Predators are a major source of stress in natural systems because their prey must balance the benefits of feeding with the risk of being eaten. Although this 'fear' of being eaten often drives the organization and dynamics of many natural systems, we know little about how such risk effects will be altered by climate change. Here, we examined the interactive consequences of predator avoidance and projected climate warming in a three-level rocky intertidal food chain. We found that both predation risk and increased air and sea temperatures suppressed the foraging of prey in the middle trophic level, suggesting that warming may further enhance the top-down control of predators on communities. Prey growth efficiency, which measures the efficiency of energy transfer between trophic levels, became negative when prey were subjected to predation risk and warming. Thus, the combined effects of these stressors may represent an important tipping point for individual fitness and the efficiency of energy transfer in natural food chains. In contrast, we detected no adverse effects of warming on the top predator and the basal resources. Hence, the consequences of projected warming may be particularly challenging for intermediate consumers residing in food chains where risk dominates predator-prey interactions. © 2014 John Wiley & Sons Ltd.

Fenton Oxidation Kinetics and Intermediates of Nonylphenol Ethoxylates

PubMed Central

Cui, Kai; Yi, Hao; Zhou, Zi-jian; Zhuo, Qiong-fang; Bing, Yong-xin; Guo, Qing-wei; Xu, Zhen-cheng

2014-01-01

Abstract Removal of nonylphenol ethoxylates (NPEOs) in aqueous solution by Fenton oxidation process was studied in a laboratory-scale batch reactor. Operating parameters, including initial pH temperature, hydrogen peroxide, and ferrous ion dosage, were thoroughly investigated. Maximum NPEOs reduction of 84% was achieved within 6 min, under an initial pH of 3.0, 25°C, an H2O2 dosage of 9.74×10−3 M, and a molar ratio of [H2O2]/[Fe2+] of 3. A modified pseudo-first-order kinetic model was found to well represent experimental results. Correlations of reaction rate constants and operational parameters were established based on experimental data. Results indicated that the Fenton oxidation rate and removal efficiency were more dependent on the dosage of H2O2 than Fe2+, and the apparent activation energy (ΔE) was 17.5 kJ/mol. High-performance liquid chromatography and gas chromatograph mass spectrometer analytical results indicated degradation of NPEOs obtained within the first 2 min stepwise occurred by ethoxyl (EO) unit shortening. Long-chain NPEOs mixture demonstrated a higher degradation rate than shorter-chain ones. Nonylphenol (NP), short-chain NPEOs, and NP carboxyethoxylates were identified as the primary intermediates, which were mostly further degraded. PMID:24868141

Intermediate-filaments: from disordered building blocks to well-ordered cells

NASA Astrophysics Data System (ADS)

Kornreich, Micha; Malka-Gibor, Eti; Laser-Azogui, Adi; Doron, Ofer; Avinery, Ram; Herrmann, Harald; Beck, Roy

In the past decade it was found that ~50% of human proteins contain long disordered regions, which play significant functional roles. As these regions lack a defined 3D folded structure, their ensemble conformations can be studied using polymer physics statistical-mechanics arguments. We measure the structure and mechanical response of hydrogels composed of neuronal intermediate filaments proteins. In the nervous system, these proteins provide cells with their mechanical support and shape, via interactions of their long, highly charged and disordered protein chains. We employ synchrotron small-angle X-ray scattering and various microscopy techniques to investigate such hydrogels from the nano- to the macro-scale. In contrast to previous polymer physics theories and experiments, we find that shorter and less charged chains can promote network expansion. The results are explained by intricate interactions between specific domains on the interacting chains, and also suggest a novel structural justification for the changing protein compositions observed during neuronal development. We address the following questions: Can protein disorder have an important role in cellular architecture? Can structural disorder in the micro-scale induce orientational and translational order on the macro-scale? How do the physical properties of disordered protein regions, such as charge, length, and hydrophobicity, modulate the cellular super-structure?

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

PubMed Central

Calderone, Christopher T.; Bumpus, Stefanie B.; Kelleher, Neil L.; Walsh, Christopher T.; Magarvey, Nathan A.

2008-01-01

The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit α-hydroxyisocaproate (α-HIC). We show here that it arises from initial activation of α-ketoisocaproate (α-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The α-KIC unit is elongated to α-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the α-keto group of α-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the α-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function α- and β-KR acting on disparate positions of a growing chain intermediate. PMID:18723688

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

Kinetics and mechanism for the sonochemical degradation of a nonionic surfactant.

PubMed

Singla, Ritu; Grieser, Franz; Ashokkumar, Muthupandian

2009-03-26

The sonolytic degradation of the nonionic surfactant, octaethylene glycol monododecyl ether (C(12)E(8)), has been studied at various initial concentrations below and above its critical micelle concentration (CMC). It has been observed that the degradation rate increases with an increase in the initial concentration of the surfactant until the CMC is reached. Above the CMC an almost constant degradation rate is observed, suggesting that the surfactant in its monomer form is involved in the degradation process. The degradation process of C(12)E(8) involves two distinct primary processes occurring at the bubble/solution interface: (a) hydroxylation/oxidation of the surfactant and (b) pyrolytic fragmentation of the surfactant. The oxidative cleavage of ethylene oxide units provides evidence for OH radical attack. Hydroxylation of the ethoxy chain gives rise to various short-chain carboxyalkyl-polyethylene glycol intermediates. The polyethylene glycol chain formed, due to the scission of the C(12)E(8) molecule, undergoes rapid hydroxylation/oxidation to yield simple compounds that have the potential to undergo further degradation. The detection of multiple intermediates indicates that several processes affect the complete degradation pathways of the surfactant molecule. TOC analysis, however, indicates that the sonolytic mineralization of the surfactant is difficult to achieve at reasonable rates due to the relatively low surface activity of the degradation products formed during sonolysis.

ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity.

PubMed

Shao, Y; Aplin, A E

2012-01-19

B-cell lymphoma 2 (Bcl-2) homology 3 (BH3)-only proteins represent a class of pro-apoptotic factors that neutralize pro-survival Bcl-2 proteins, and, in some cases, directly activate Bax. The mechanisms of control and the role of BH3-only proteins, such as Bcl-2 like protein 11 extra large and Bad are well studied. By contrast, relatively little is known about the regulation and role of Bcl-2 modifying factor (Bmf). The B-RAF oncogene is mutated in ∼8% of human tumors. We have previously shown that Bmf is upregulated at the transcript level and is required for apoptosis induced by targeting B-RAF signaling in tumor cells harboring mutant B-RAF. In this study, we show that Bmf is regulated at the post-translational level by mutant B-RAF-MEK-ERK2 signaling. Extracellular signal-regulated kinase (ERK2) directly phosphorylates Bmf on serine 74 and serine 77 residues with serine 77 being the predominant site. In addition, serine 77 phosphorylation reduces Bmf pro-apoptotic activity likely through a mechanism independent of altering Bmf localization to the mitochondria and/or interactions with dynein light chain 2 and the pro-survival proteins, B-cell lymphoma extra large, Bcl-2 and Mcl-1. These data identify a novel mode of regulation in Bmf that modulates its pro-apoptotic activity in mutant B-RAF tumor cells.

Endogenous association of Bim BH3-only protein with Mcl-1, Bcl-xL and Bcl-2 on mitochondria in human B cells.

PubMed

Gomez-Bougie, Patricia; Bataille, Régis; Amiot, Martine

2005-03-01

Bim is an essential regulator of lymphoid system homeostasis and appears essential for B cell apoptosis induction. The mechanism by which Bim isoforms are held in an inactive form remains poorly documented in normal B cells. In the current study, we demonstrated that in normal tonsil B cells the three major Bim isoforms are strongly associated with the anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-x(L). On the other hand, only a weak association of BimEL and L with the dynein LC8 chain has been found. In addition, there is no free Bim in normal B cells. Moreover, subcellular fractionation demonstrated that Bim and the anti-apoptotic counterparts are localized preferentially in the mitochondria-rich fraction. The fact that most Bim was found in this fraction supports the hypothesis that it is sequestered by anti-apoptotic molecules in mitochondria where its pro-apoptotic activity is controlled. Of interest, BimS is essentially complexed to Mcl-1 and the Mcl-1/Bim complex is the most abundant among the three types of complexes. This supports the idea that this complex is critical for the control of B cell death. In conclusion, these results favor a model in which Bim release from anti-apoptotic proteins is a critical event for initiation of apoptosis.

The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

PubMed Central

Jurado, Sabine; Gleeson, Kimberly; O’Donnell, Kristy; Izon, David J.; Walkley, Carl R.; Strasser, Andreas; Tarlinton, David M.

2012-01-01

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis. PMID:22891272

Analytical model of multi-planetary resonant chains and constraints on migration scenarios

NASA Astrophysics Data System (ADS)

Delisle, J.-B.

2017-09-01

Resonant chains are groups of planets for which each pair is in resonance, with an orbital period ratio locked at a rational value (2/1, 3/2, etc.). Such chains naturally form as a result of convergent migration of the planets in the proto-planetary disk. In this article, I present an analytical model of resonant chains of any number of planets. Using this model, I show that a system captured in a resonant chain can librate around several possible equilibrium configurations. The probability of capture around each equilibrium depends on how the chain formed, and especially on the order in which the planets have been captured in the chain. Therefore, for an observed resonant chain, knowing around which equilibrium the chain is librating allows for constraints to be put on the formation and migration scenario of the system. I apply this reasoning to the four planets orbiting Kepler-223 in a 3:4:6:8 resonant chain. I show that the system is observed around one of the six equilibria predicted by the analytical model. Using N-body integrations, I show that the most favorable scenario to reproduce the observed configuration is to first capture the two intermediate planets, then the outermost, and finally the innermost.

Spontaneous oscillation and fluid-structure interaction of cilia.

PubMed

Han, Jihun; Peskin, Charles S

2018-04-24

The exact mechanism to orchestrate the action of hundreds of dynein motor proteins to generate wave-like ciliary beating remains puzzling and has fascinated many scientists. We present a 3D model of a cilium and the simulation of its beating in a fluid environment. The model cilium obeys a simple geometric constraint that arises naturally from the microscopic structure of a real cilium. This constraint allows us to determine the whole 3D structure at any instant in terms of the configuration of a single space curve. The tensions of active links, which model the dynein motor proteins, follow a postulated dynamical law, and together with the passive elasticity of microtubules, this dynamical law is responsible for the ciliary motions. In particular, our postulated tension dynamics lead to the instability of a symmetrical steady state, in which the cilium is straight and its active links are under equal tensions. The result of this instability is a stable, wave-like, limit cycle oscillation. We have also investigated the fluid-structure interaction of cilia using the immersed boundary (IB) method. In this setting, we see not only coordination within a single cilium but also, coordinated motion, in which multiple cilia in an array organize their beating to pump fluid, in particular by breaking phase synchronization.

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

PubMed

Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

2010-10-06

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Variations in the ultrastructure of human nasal cilia including abnormalities found in retinitis pigmentosa.

PubMed Central

Fox, B; Bull, T B; Arden, G B

1980-01-01

The electron microscopic structure of cilia from the inferior turbinate of the nose was studied in 12 adults, four with chronic sinusitis, one with allergic rhinitis, two with bronchiectasis, three with deviated nasal septum, and two normals. The changes are compared with those found in nasal cilia in 14 patients with retinitis pigmentosa. There were compound cilia in the seven cases with chronic sinusitis, allergic rhinitis, and bronchiectasis but, apart from this, the structure of the cilia was similar in all 12 cases. There were variations in the microtubular pattern in about 4% of cilia, dynein arms were not seen in 4%, and in the rest an average of 5-6 dynein arms were seen in each cilium. The orientation of the cilia was 0 to 90 degrees. In the retinitis pigmentosa patients there was a highly significant increase in cilial abnormalities. The establishment on a quantitative basis of the variations in normal structure of nasal cilila facilitated the recognition of an association between cilial abnormalities and retinitis pigmentosa and should help in the identification of associations that may exist between cilial abnormalities and other diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 Fig. 8 PMID:7400333

Assessing the Impact of Electrostatic Drag on Processive Molecular Motor Transport.

PubMed

Smith, J Darby; McKinley, Scott A

2018-06-04

The bidirectional movement of intracellular cargo is usually described as a tug-of-war among opposite-directed families of molecular motors. While tug-of-war models have enjoyed some success, recent evidence suggests underlying motor interactions are more complex than previously understood. For example, these tug-of-war models fail to predict the counterintuitive phenomenon that inhibiting one family of motors can decrease the functionality of opposite-directed transport. In this paper, we use a stochastic differential equations modeling framework to explore one proposed physical mechanism, called microtubule tethering, that could play a role in this "co-dependence" among antagonistic motors. This hypothesis includes the possibility of a trade-off: weakly bound trailing molecular motors can serve as tethers for cargoes and processing motors, thereby enhancing motor-cargo run lengths along microtubules; however, this introduces a cost of processing at a lower mean velocity. By computing the small- and large-time mean-squared displacement of our theoretical model and comparing our results to experimental observations of dynein and its "helper protein" dynactin, we find some supporting evidence for microtubule tethering interactions. We extrapolate these findings to predict how dynein-dynactin might interact with the opposite-directed kinesin motors and introduce a criterion for when the trade-off is beneficial in simple systems.

Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

PubMed

Mitchell, V; Sigala, J; Ballot, C; Jumeau, F; Barbotin, A L; Duhamel, A; Rives, N; Rigot, J M; Escalier, D; Peers, M C

2015-03-01

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk. © 2014 Blackwell Verlag GmbH.

Simultaneous and spectroscopic redox molecular imaging of multiple free radical intermediates using dynamic nuclear polarization-magnetic resonance imaging.

PubMed

Hyodo, Fuminori; Ito, Shinji; Yasukawa, Keiji; Kobayashi, Ryoma; Utsumi, Hideo

2014-08-05

Redox reactions that generate free radical intermediates are essential to metabolic processes. However, their intermediates can produce reactive oxygen species, which may promote diseases related to oxidative stress. We report here the use of dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) to conduct redox molecular imaging. Using DNP-MRI, we obtained simultaneous images of free radical intermediates generated from the coenzyme Q10 (CoQ10), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) involved in the mitochondrial electron transport chain as well as the radicals derived from vitamins E and K1. Each of these free radicals was imaged in real time in a phantom comprising a mixture of free radicals localized in either lipophilic or aqueous environments. Changing the frequency of electron spin resonance (ESR) irradiation also allowed each of the radical species to be distinguished in the spectroscopic images. This study is the first to report the spectroscopic DNP-MRI imaging of free radical intermediates that are derived from endogenous species involved in metabolic processes.

Hole hopping through tyrosine/tryptophan chains protects proteins from oxidative damage

PubMed Central

Gray, Harry B.; Winkler, Jay R.

2015-01-01

Living organisms have adapted to atmospheric dioxygen by exploiting its oxidizing power while protecting themselves against toxic side effects. Reactive oxygen and nitrogen species formed during oxidative stress, as well as high-potential reactive intermediates formed during enzymatic catalysis, could rapidly and irreversibly damage polypeptides were protective mechanisms not available. Chains of redox-active tyrosine and tryptophan residues can transport potentially damaging oxidizing equivalents (holes) away from fragile active sites and toward protein surfaces where they can be scavenged by cellular reductants. Precise positioning of these chains is required to provide effective protection without inhibiting normal function. A search of the structural database reveals that about one third of all proteins contain Tyr/Trp chains composed of three or more residues. Although these chains are distributed among all enzyme classes, they appear with greatest frequency in the oxidoreductases and hydrolases. Consistent with a redox-protective role, approximately half of the dioxygen-using oxidoreductases have Tyr/Trp chain lengths ≥3 residues. Among the hydrolases, long Tyr/Trp chains appear almost exclusively in the glycoside hydrolases. These chains likely are important for substrate binding and positioning, but a secondary redox role also is a possibility. PMID:26195784

Effect of chain stiffness on the competition between crystallization and glass-formation in model unentangled polymers

NASA Astrophysics Data System (ADS)

Nguyen, Hong T.; Smith, Tyler B.; Hoy, Robert S.; Karayiannis, Nikos Ch.

2015-10-01

We map out the solid-state morphologies formed by model soft-pearl-necklace polymers as a function of chain stiffness, spanning the range from fully flexible to rodlike chains. The ratio of Kuhn length to bead diameter (lK/r0) increases monotonically with increasing bending stiffness kb and yields a one-parameter model that relates chain shape to bulk morphology. In the flexible limit, monomers occupy the sites of close-packed crystallites while chains retain random-walk-like order. In the rodlike limit, nematic chain ordering typical of lamellar precursors coexists with close-packing. At intermediate values of bending stiffness, the competition between random-walk-like and nematic chain ordering produces glass-formation; the range of kb over which this occurs increases with the thermal cooling rate | T ˙ | implemented in our molecular dynamics simulations. Finally, values of kb between the glass-forming and rodlike ranges produce complex ordered phases such as close-packed spirals. Our results should provide a useful initial step in a coarse-grained modeling approach to systematically determining the effect of chain stiffness on the crystallization-vs-glass-formation competition in both synthetic and colloidal polymers.

An analytical approach to top predator interference on the dynamics of a food chain model

NASA Astrophysics Data System (ADS)

Senthamarai, R.; Vijayalakshmi, T.

2018-04-01

In this paper, a nonlinear mathematical model is proposed and analyzed to study of top predator interference on the dynamics of a food chain model. The mathematical model is formulated using the system of non-linear ordinary differential equations. In this model, there are three state dimensionless variables, viz, size of prey population x, size of intermediate predator y and size of top predator population z. The analytical results are compared with the numerical simulation using MATLAB software and satisfactory results are noticed.

Teaching Probability in Intermediate Grades

ERIC Educational Resources Information Center

Engel, Arthur

1971-01-01

A discussion of the importance and procedures for including probability in the elementary through secondary mathematics curriculum is presented. Many examples and problems are presented which the author feels students can understand and will be motivated to do. Random digits, Monte Carlo methods, combinatorial theory, and Markov chains are…

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

PubMed Central

Betts, S. D.; King, J.

1998-01-01

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly. PMID:9684883

Hard-X-Ray-Induced Multistep Ultrafast Dissociation

NASA Astrophysics Data System (ADS)

Travnikova, Oksana; Marchenko, Tatiana; Goldsztejn, Gildas; Jänkälä, Kari; Sisourat, Nicolas; Carniato, Stéphane; Guillemin, Renaud; Journel, Loïc; Céolin, Denis; Püttner, Ralph; Iwayama, Hiroshi; Shigemasa, Eiji; Piancastelli, Maria Novella; Simon, Marc

2016-05-01

Creation of deep core holes with very short (τ ≤1 fs ) lifetimes triggers a chain of relaxation events leading to extensive nuclear dynamics on a few-femtosecond time scale. Here we demonstrate a general multistep ultrafast dissociation on an example of HCl following Cl 1 s →σ* excitation. Intermediate states with one or multiple holes in the shallower core electron shells are generated in the course of the decay cascades. The repulsive character and large gradients of the potential energy surfaces of these intermediates enable ultrafast fragmentation after the absorption of a hard x-ray photon.

Substrate degradation by the proteasome: a single-molecule kinetic analysis

PubMed Central

Lu, Ying; Lee, Byung-hoon; King, Randall W; Finley, Daniel; Kirschner, Marc W

2015-01-01

To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation. PMID:25859050

Wave propagation characteristics of a magnetic granular chain

NASA Astrophysics Data System (ADS)

Leng, Dingxin; Liu, Guijie; Sun, Lingyu; Wang, Xiaojie

2017-10-01

We investigate the wave propagation characteristics of a horizontal alignment of magnetic grains under a non-uniform magnetic field. The magnetic force of each grain is obtained using Maxwell's principle. The contact interaction of grains is based on Hertz potential. The effects of magnetic field strength on the dynamic responses of a granular chain under strong, intermediate, and weak amplitudes of incident impulses in comparison with static precompression force are studied. Different wave propagation modes induced by the magnetic field are observed. The applied field strength demonstrably reinforces the granular-position-dependent behaviors of decreasing amplitude and increasing wave propagation velocity. The magnetic field-induced features of a magnetic granular chain have potential applications in adaptive structures for shock attenuation.

Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

USDA-ARS?s Scientific Manuscript database

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by the yeast, particularly when the carbon source is acid-treated lignocell...

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

ANAEROBIC BIODEGRADATION OF VEGETABLE OIL AND ITS METABOLIC INTERMEDIATES IN OIL-ENRICHED FRESHWATER SEDIMENTS

EPA Science Inventory

Anaerobic biodegradation of vegetable oil in freshwater sediments is strongly inhibited by high concentrations of oil, but the presence of ferric hydroxide relieves the inhibition. The effect of ferric hydroxide is not due to physical or chemical interactions with long-chain fatt...

Selective Hydrodeoxygenation of Vegetable Oils and Waste Cooking Oils to Green Diesel Using a Silica-Supported Ir-ReOx Bimetallic Catalyst.

PubMed

Liu, Sibao; Simonetti, Trent; Zheng, Weiqing; Saha, Basudeb

2018-05-09

High yields of diesel-range alkanes are prepared by hydrodeoxygenation of vegetable oils and waste cooking oils over ReO x -modified Ir/SiO 2 catalysts under mild reaction conditions. The catalyst containing a Re/Ir molar ratio of 3 exhibits the best performance, achieving 79-85 wt % yield of diesel-range alkanes at 453 K and 2 MPa H 2 . The yield is nearly quantitative for the theoretical possible long-chain alkanes on the basis of weight of the converted oils. The catalyst retains comparable activity upon regeneration through calcination. Control experiments using probe molecules as model substrates suggest that C=C bonds of unsaturated triglycerides and free fatty acids are first hydrogenated to their corresponding saturated intermediates, which are then converted to aldehyde intermediates through hydrogenolysis of acyl C-O bonds and subsequently hydrogenated to fatty alcohols. Finally, long-chain alkanes without any carbon loss are formed by direct hydrogenolysis of the fatty alcohols. Small amounts of alkanes with one carbon fewer are also formed by decarbonylation of the aldehyde intermediates. A synergy between Ir and partially reduced ReO x sites is discussed to elucidate the high activity of Ir-ReO x /SiO 2. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Hydrophobic Interactions of Polymers with Water and Phospholipid Membranes Using Molecular Dynamics Simulations

NASA Astrophysics Data System (ADS)

Drenscko, Mihaela

Polymers and lipid membranes are both essential soft materials. The structure and hydrophobicity/hydrophilicity of polymers, as well as the solvent they are embedded in, ultimately determines their size and shape. Understating the variation of shape of the polymer as well as its interactions with model biological membranes can assist in understanding the biocompatibility of the polymer itself. Computer simulations, in particular molecular dynamics, can aid in characterization of the interaction of polymers with solvent, as well as polymers with model membranes. In this thesis, molecular dynamics serve to describe polymer interactions with a solvent (water) and with a lipid membrane. To begin with, we characterize the hydrophobic collapse of single polystyrene chains in water using molecular dynamics simulations. Specifically, we calculate the potential of mean force for the collapse of a single polystyrene chain in water using metadynamics, comparing the results between all atomistic with coarse-grained molecular simulation. We next explore the scaling behavior of the collapsed globular shape at the minimum energy configuration, characterized by the radius of gyration, as a function of chain length. The exponent is close to one third, consistent with that predicted for a polymer chain in bad solvent. We also explore the scaling behavior of the Solvent Accessible Surface Area (SASA) as a function of chain length, finding a similar exponent for both all-atomistic and coarse-grained simulations. Furthermore, calculation of the local water density as a function of chain length near the minimum energy configuration suggests that intermediate chain lengths are more likely to form dewetted states, as compared to shorter or longer chain lengths. Next, in order to investigate the molecular interactions between single hydrophobic polymer chains and lipids in biological membranes and at lipid membrane/solvent interface, we perform a series of molecular dynamics simulations of small membranes using all atomistic and coarse-grained methods. The molecular interaction between common polymer chains used in biomedical applications and the cell membrane is unknown. This interaction may affect the biocompatibility of the polymer chains. Molecular dynamics simulations offer an emerging tool to characterize the interaction between common degradable polymer chains used in biomedical applications, such as polycaprolactone, and model cell membranes. We systematically characterize with long-time all-atomistic molecular dynamics simulations the interaction between single polycaprolactone chains of varying chain lengths with a model phospholipid membrane. We find that the length of polymer chain greatly affects the nature of interaction with the membrane, as well as the membrane properties. Furthermore, we next utilize advanced sampling techniques in molecular dynamics to characterize the two-dimensional free energy surface for the interaction of varying polymer chain lengths (short, intermediate, and long) with model cell membranes. We find that the free energy minimum shifts from the membrane-water interface to the hydrophobic core of the phospholipid membrane as a function of chain length. These results can be used to design polymer chain lengths and chemistries to optimize their interaction with cell membranes at the molecular level.

Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

PubMed

Burghardt, Thomas P; Sikkink, Laura A

2013-02-19

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments

PubMed Central

Nishimura, Chiaki; Dyson, H. Jane; Wright, Peter E.

2005-01-01

Hydrogen/deuterium exchange followed by trapping of the labeled species in the aprotic solvent DMSO has been used to elucidate structure in both the burst-phase molten globule-folding intermediate of apomyoglobin and in an equilibrium intermediate that models the kinetic intermediate. Precise estimates can be made of exchange times in an interrupted exchange-out experiment at pH 4 followed by analysis in DMSO solution, giving extensive sequence-specific information about the structure of the equilibrium intermediate. In addition, the use of DMSO as a solvent for NMR measurements after quench-flow pH-pulse labeling experiments gives a greatly increased data set for the elucidation of the kinetic folding pathway. Interestingly, differences are observed in some regions of apomyoglobin between the equilibrium and kinetic intermediates. These differences are quantitative rather than qualitative; that is, the overall patterns of labeling and secondary structure formation remain similar between the two species. However, local differences are observed, which probably reflect the difference in the solution conditions for the equilibrium experiment (pH 4) vs. the kinetic experiment (pH 6) and the change in the status of the stabilizing hydrogen bond between the side chains of His-24 and His-119. PMID:15769860

Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride

PubMed Central

Roe, Charles R.; Sweetman, Lawrence; Roe, Diane S.; David, France; Brunengraber, Henri

2002-01-01

The current dietary treatment of long-chain fatty acid oxidation defects (high carbohydrate with medium-even-chain triglycerides and reduced amounts of long-chain fats) fails, in many cases, to prevent cardiomyopathy, rhabdomyolysis, and muscle weakness. We hypothesized that the apparent defect in energy production results from a depletion of the catalytic intermediates of the citric acid cycle via leakage through cell membranes (cataplerosis). We further hypothesized that replacing dietary medium-even-chain fatty acids (precursors of acetyl-CoA) by medium-odd-chain fatty acids (precursors of acetyl-CoA and anaplerotic propionyl-CoA) would restore energy production and improve cardiac and skeletal muscle function. We fed subjects with long-chain defects a controlled diet in which the fat component was switched from medium-even-chain triglycerides to triheptanoin. In three patients with very-long-chain acyl-CoA dehydrogenase deficiency, this treatment led rapidly to clinical improvement that included the permanent disappearance of chronic cardiomyopathy, rhabdomyolysis, and muscle weakness (for more than 2 years in one child), and of rhabdomyolysis and weakness in the others. There was no evidence of propionyl overload in these patients. The treatment has been well tolerated for up to 26 months and opens new avenues for the management of patients with mitochondrial fat oxidation disorders. PMID:12122118

Kinetic aspects of chain growth in Fischer-Tropsch synthesis.

PubMed

Filot, Ivo A W; Zijlstra, Bart; Broos, Robin J P; Chen, Wei; Pestman, Robert; Hensen, Emiel J M

2017-04-28

Microkinetics simulations are used to investigate the elementary reaction steps that control chain growth in the Fischer-Tropsch reaction. Chain growth in the FT reaction on stepped Ru surfaces proceeds via coupling of CH and CR surface intermediates. Essential to the growth mechanism are C-H dehydrogenation and C hydrogenation steps, whose kinetic consequences have been examined by formulating two novel kinetic concepts, the degree of chain-growth probability control and the thermodynamic degree of chain-growth probability control. For Ru the CO conversion rate is controlled by the removal of O atoms from the catalytic surface. The temperature of maximum CO conversion rate is higher than the temperature to obtain maximum chain-growth probability. Both maxima are determined by Sabatier behavior, but the steps that control chain-growth probability are different from those that control the overall rate. Below the optimum for obtaining long hydrocarbon chains, the reaction is limited by the high total surface coverage: in the absence of sufficient vacancies the CHCHR → CCHR + H reaction is slowed down. Beyond the optimum in chain-growth probability, CHCR + H → CHCHR and OH + H → H 2 O limit the chain-growth process. The thermodynamic degree of chain-growth probability control emphasizes the critical role of the H and free-site coverage and shows that at high temperature, chain depolymerization contributes to the decreased chain-growth probability. That is to say, during the FT reaction chain growth is much faster than chain depolymerization, which ensures high chain-growth probability. The chain-growth rate is also fast compared to chain-growth termination and the steps that control the overall CO conversion rate, which are O removal steps for Ru.

Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates.

PubMed

Chamani, J; Moosavi-Movahedi, A A; Rajabi, O; Gharanfoli, M; Momen-Heravi, M; Hakimelahi, G H; Neamati-Baghsiah, A; Varasteh, A R

2006-01-01

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of beta-lactoglobulin was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at special condition. The effect of n-alkyl sulfates on the structure of native beta-lactoglobulin at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of non-native alpha-helical intermediate. The addition of various concentrations of n-alkyl sulfates to the native state of beta-lactoglobulin (pH 2) appears to support the stabilized form of non-native alpha-helical intermediate at pH 2. The m values of the intermediate state of beta-lactoglobulin by SOS, SDeS, SDS and STS showed substantial variation. The enhancement of m values as the stability criterion of non-native alpha-helical intermediate state corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the non-native alpha-helical intermediate state.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

PubMed Central

Butardo, Vito M.; Fitzgerald, Melissa A.; Bird, Anthony R.; Gidley, Michael J.; Flanagan, Bernadine M.; Larroque, Oscar; Resurreccion, Adoracion P.; Laidlaw, Hunter K. C.; Jobling, Stephen A.; Morell, Matthew K.; Rahman, Sadequr

2011-01-01

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed. PMID:21791436

Application of pulsed-field gel electrophoresis to characterise and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility.

PubMed

Mullane, N R; Whyte, P; Wall, P G; Quinn, T; Fanning, S

2007-05-01

Enterobacter sakazakii (E. sakazakii) contamination of powdered infant formula (PIF) and its processing environment was monitored between April 2005 and March 2006. The purpose of the monitoring programme was to locate points of contamination, investigate clonal persistence, and identify possible dissemination routes along the processing chain. A total of 80 E. sakazakii isolates were recovered from the manufacturing facility. The overall frequency of isolation of E. sakazakii in intermediate and final product was 2.5%, while specific locations in the processing environment were contaminated at frequencies up to 31%. All E. sakazakii isolates were characterised by pulsed-field gel electrophoresis (PFGE). XbaI macrorestriction digests yielded 19 unique pulse-types that could be grouped into 6 clusters of between 5 and 32 isolates. The formation of large clusters was consistent with the presence of a number of clones in the manufacturing environment. While the majority of isolates were of environmental origin (72.5%), no cluster was confined to one specific location and indistinguishable PFGE profiles were generated from isolates cultured from the manufacturing environment, sampling points along the processing chain and from intermediate and final product. These findings suggest that the manufacturing environment serves as a key route for sporadic contamination of PIF. These data will support the development of efficient intervention measures contributing to the reduction of E. sakazakii in the PIF processing chain.

Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain.

PubMed

Kirikoshi, Ryota; Manabe, Noriyoshi; Takahashi, Ohgi

2017-02-16

The Asn-Gly-Arg (NGR) motif and its deamidation product iso Asp-Gly-Arg ( iso DGR) have recently attracted considerable attention as tumor-targeting ligands. Because an NGR-containing peptide and the corresponding iso DGR-containing peptide target different receptors, the spontaneous NGR deamidation can be used in dual targeting strategies. It is well known that the Asn deamidation proceeds via a succinimide derivative. In the present study, we computationally investigated the mechanism of succinimide formation from a cyclic peptide, c[CH₂CO-NGRC]-NH₂, which has recently been shown to undergo rapid deamidation in a phosphate buffer. An H₂PO₄ - ion was explicitly included in the calculations. We employed the density functional theory using the B3LYP functional. While geometry optimizations were performed in the gas phase, hydration Gibbs energies were calculated by the SM8 (solvation model 8) continuum model. We have found a pathway leading to the five-membered ring tetrahedral intermediate in which both the H₂PO₄ - ion and the Arg side chain act as catalyst. This intermediate, once protonated at the NH₂ group on the five-membered ring, was shown to easily undergo NH₃ elimination leading to the succinimide formation. This study is the first to propose a possible catalytic role for the Arg side chain in the NGR deamidation.

Determining Role of the Chain Mechanism in the Temperature Dependence of the Gas-Phase Rate of Combustion Reactions

NASA Astrophysics Data System (ADS)

Azatyan, V. V.; Bolod'yan, I. A.; Kopylov, N. P.; Kopylov, S. N.; Prokopenko, V. M.; Shebeko, Yu. N.

2018-05-01

It is shown that the strong dependence of the rate of gas-phase combustion reactions on temperature is determined by the high values of the reaction rate constants of free atoms and radicals. It is established that with a branched chain mechanism, a special role in the reaction rate temperature dependence is played by positive feedback between the concentrations of active intermediate species and the rate of their change. The role of the chemical mechanism in the temperature dependence of the process rate with and without inhibitors is considered.

Enzymes involved in branched-chain amino acid metabolism in humans.

PubMed

Adeva-Andany, María M; López-Maside, Laura; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Sixto-Leal, Cristina

2017-06-01

Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.

Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L

NASA Astrophysics Data System (ADS)

Maity, Hiranmay; Reddy, Govardhan

2018-04-01

Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

Wormlike Chain Theory and Bending of Short DNA

NASA Astrophysics Data System (ADS)

Mazur, Alexey K.

2007-05-01

The probability distributions for bending angles in double helical DNA obtained in all-atom molecular dynamics simulations are compared with theoretical predictions. The computed distributions remarkably agree with the wormlike chain theory and qualitatively differ from predictions of the subelastic chain model. The computed data exhibit only small anomalies in the apparent flexibility of short DNA and cannot account for the recently reported AFM data. It is possible that the current atomistic DNA models miss some essential mechanisms of DNA bending on intermediate length scales. Analysis of bent DNA structures reveal, however, that the bending motion is structurally heterogeneous and directionally anisotropic on the length scales where the experimental anomalies were detected. These effects are essential for interpretation of the experimental data and they also can be responsible for the apparent discrepancy.

The Cognitive Architecture for Chaining of Two Mental Operations

ERIC Educational Resources Information Center

Sackur, Jerome; Dehaene, Stanislas

2009-01-01

A simple view, which dates back to Turing, proposes that complex cognitive operations are composed of serially arranged elementary operations, each passing intermediate results to the next. However, whether and how such serial processing is achieved with a brain composed of massively parallel processors, remains an open question. Here, we study…

Random Breakage of a Rod into Unit Lengths

ERIC Educational Resources Information Center

Gani, Joe; Swift, Randall

2011-01-01

In this article we consider the random breakage of a rod into "L" unit elements and present a Markov chain based method that tracks intermediate breakage configurations. The probability of the time to final breakage for L = 3, 4, 5 is obtained and the method is shown to extend in principle, beyond L = 5.

How-to-Do-It: A Physical Model Illustrating Protein Synthesis on the Ribosome.

ERIC Educational Resources Information Center

Rogerson, Allen C.; Cheney, Richard W., Jr.

1989-01-01

Describes a way to help students grasp intermediate steps in the movement and relationships of the various components involved in the addition of an amino acid to a nascent peptide chain. Includes drawings of the model in operation, construction details, and suggested shapes and labeling of components. (RT)

The p25 Subunit of the Dynactin Complex is Required for Dynein-Early Endosome Interaction

DTIC Science & Technology

2011-01-01

Recently, spectrin mutations in Drosophila melanogaster have been shown to also cause defective axonal transport and neu- ronal degeneration...A and study showed that in Arp1-RNAi–treated Drosophila S2 cells, although dynactin cannot be fully assembled and vesicle transport is defective...Muresan et al., 2001; Schroer 2004). However, this idea has been under debate recently, mainly because results from a study in Drosophila S2 cells

Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

PubMed Central

Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W.; Wainman, Alan

2015-01-01

Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs. PMID:26020779

Dynein-Dependent Transport of nanos RNA in Drosophila Sensory Neurons Requires Rumpelstiltskin and the Germ Plasm Organizer Oskar

PubMed Central

Xu, Xin; Brechbiel, Jillian L.

2013-01-01

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts. PMID:24027279

Dynein-dependent transport of nanos RNA in Drosophila sensory neurons requires Rumpelstiltskin and the germ plasm organizer Oskar.

PubMed

Xu, Xin; Brechbiel, Jillian L; Gavis, Elizabeth R

2013-09-11

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts.

FBXL5 interacts with p150 {sup Glued} and regulates its ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Ning; Liu Jing; Ding Xia

2007-07-20

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. p150 {sup Glued} is the dynactin subunit responsible for binding to dynein and microtubules. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which governs phosphorylation-dependent ubiquitination and subsequent proteolysis. Our recent study showed that the proteolysis of mitotic kinesin CENP-E is mediated by SCF via a direct Skp1 link [D. Liu, N. Zhang, J. Du, X. Cai, M. Zhu, C. Jin, Z. Dou, C. Feng, Y. Yang, L. Liu, K. Takeyasu, W. Xie, X. Yao,more » Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis, Biochem. Biophys. Res. Commun. 345 (2006) 394-402]. Here we show that F-box protein FBXL5 interacts with p150 {sup Glued} and orchestrates its turnover via ubiquitination. FBXL5 binds to p150 {sup Glued} in vitro and in vivo. FBXL5 and p150 {sup Glued} co-localize primarily in the cytoplasm with peri-nuclear enrichment in HeLa cells. Overexpression of FBXL5 promotes poly-ubiquitination of p150 {sup Glued} and protein turnover of p150 {sup Glued} . Our findings provide a potential mechanism by which p150 {sup Glued} protein function is regulated by SCFs.« less

Generation of a three-dimensional ultrastructural model of human respiratory cilia.

PubMed

Burgoyne, Thomas; Dixon, Mellisa; Luther, Pradeep; Hogg, Claire; Shoemark, Amelia

2012-12-01

The ultrastructures of cilia and flagella are highly similar and well conserved through evolution. Consequently, Chlamydomonas is commonly used as a model organism for the study of human respiratory cilia. Since detailed models of Chlamydomonas axonemes were generated using cryoelectron tomography, disparities among some of the ultrastructural features have become apparent when compared with human cilia. Extrapolating information on human disease from the Chlamydomonas model may lead to discrepancies in translational research. This study aimed to establish the first three-dimensional ultrastructural model of human cilia. Tomograms of transverse sections (n = 6) and longitudinal sections (n = 9) of human nasal respiratory cilia were generated from three healthy volunteers. Key features of the cilium were resolved using subatomic averaging, and were measured. For validation of the method, a model of the well characterized structure of Chlamydomonas reinhardtii was simultaneously generated. Data were combined to create a fully quantified three-dimensional reconstruction of human nasal respiratory cilia. We highlight key differences in the axonemal sheath, microtubular doublets, radial spokes, and dynein arms between the two structures. We show a decreased axial periodicity of the radial spokes, inner dynein arms, and central pair protrusions in the human model. We propose that this first human model will provide a basis for research into the function and structure of human respiratory cilia in health and in disease.

A role for microtubules in sorting endocytic vesicles in rat hepatocytes.

PubMed Central

Goltz, J S; Wolkoff, A W; Novikoff, P M; Stockert, R J; Satir, P

1992-01-01

The vectorial nature of hepatocyte receptor-mediated endocytosis (RME) and its susceptibility to cytoskeletal disruptors has suggested that a polarized network of microtubules plays a vital role in directed movement during sorting. Using as markers a well-known ligand, asialoorosomucoid, and its receptor, we have isolated endocytic vesicles that bind directly to and interact with stabilized endogenous hepatocyte microtubules at specific times during a synchronous, experimentally initiated, single wave of RME. Both ligand- and receptor-containing vesicles copelleted with microtubules in the absence of ATP but did not pellet under similar conditions when microtubules were not polymerized. When 5 mM ATP was added to preparations of microtubule-bound vesicles, ligand-containing vesicles were released into the supernatant, while receptor-containing vesicles remained immobilized on the microtubules. Release of ligand-containing vesicles from microtubules was prevented by monensin treatment during the endocytic wave. Several proteins, including the microtubule motor protein cytoplasmic dynein, were present in these preparations and were released from microtubule pellets by ATP addition concomitantly with ligand. These results suggest that receptor domains within the endosome can be immobilized by attachment to microtubules so that, following monensin-sensitive dissociation of ligand from receptor, ligand-containing vesicles can be pulled along microtubules away from the receptor domains by a motor molecule, such as cytoplasmic dynein, thereby delineating sorting. Images PMID:1353884

Long-chain fatty acid combustion rate is associated with unique metabolite profiles in skeletal muscle mitochondria.

PubMed

Seifert, Erin L; Fiehn, Oliver; Bezaire, Véronic; Bickel, David R; Wohlgemuth, Gert; Adams, Sean H; Harper, Mary-Ellen

2010-03-24

Incomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA beta-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate. Large-scale unbiased metabolomics analysis was performed using GC/TOF-MS on buffer and mitochondrial matrix fractions obtained prior to and after 20 min of palmitate catabolism (n = 7 mice/condition). Three palmitate concentrations (2, 9 and 19 microM; corresponding to low, intermediate and high oxidation rates) and 9 microM palmitate plus tricarboxylic acid (TCA) cycle and electron transport chain inhibitors were each tested and compared to zero palmitate control incubations. Paired comparisons of the 0 and 20 min samples were made by Student's t-test. False discovery rate were estimated and Type I error rates assigned. Major metabolite groups were organic acids, amines and amino acids, free fatty acids and sugar phosphates. Palmitate oxidation was associated with unique profiles of metabolites, a subset of which correlated to palmitate oxidation rate. In particular, palmitate oxidation rate was associated with distinct changes in the levels of TCA cycle intermediates within and effluxed from mitochondria. This proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle.

Long-Chain Fatty Acid Combustion Rate Is Associated with Unique Metabolite Profiles in Skeletal Muscle Mitochondria

PubMed Central

Seifert, Erin L.; Fiehn, Oliver; Bezaire, Véronic; Bickel, David R.; Wohlgemuth, Gert; Adams, Sean H.; Harper, Mary-Ellen

2010-01-01

Background/Aim Incomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA β-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate. Methodology/Principal Findings Large-scale unbiased metabolomics analysis was performed using GC/TOF-MS on buffer and mitochondrial matrix fractions obtained prior to and after 20 min of palmitate catabolism (n = 7 mice/condition). Three palmitate concentrations (2, 9 and 19 µM; corresponding to low, intermediate and high oxidation rates) and 9 µM palmitate plus tricarboxylic acid (TCA) cycle and electron transport chain inhibitors were each tested and compared to zero palmitate control incubations. Paired comparisons of the 0 and 20 min samples were made by Student's t-test. False discovery rate were estimated and Type I error rates assigned. Major metabolite groups were organic acids, amines and amino acids, free fatty acids and sugar phosphates. Palmitate oxidation was associated with unique profiles of metabolites, a subset of which correlated to palmitate oxidation rate. In particular, palmitate oxidation rate was associated with distinct changes in the levels of TCA cycle intermediates within and effluxed from mitochondria. Conclusions/Significance This proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle. PMID:20352092

Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols

PubMed Central

Avalos, José L.; Fink, Gerald R.; Stephanopoulos, Gregory

2013-01-01

Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeted expression of metabolic pathways to mitochondria can increase production levels compared with expression of the same pathways in the cytoplasm. Compartmentalisation of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overexpressing only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains reveals that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves higher local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion, and reducing the loss of intermediates to competing pathways. PMID:23417095

Depletion force induced collective motion of microtubules driven by kinesin

NASA Astrophysics Data System (ADS)

Inoue, Daisuke; Mahmot, Bulbul; Kabir, Arif Md. Rashedul; Farhana, Tamanna Ishrat; Tokuraku, Kiyotaka; Sada, Kazuki; Konagaya, Akihiko; Kakugo, Akira

2015-10-01

Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects.Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02213d

Topological frustration in βα-repeat proteins: sequence diversity modulates the conserved folding mechanisms of α/β/α sandwich proteins

PubMed Central

Hills, Ronald D.; Kathuria, Sagar V.; Wallace, Louise A.; Day, Iain J.; Brooks, Charles L.; Matthews, C. Robert

2010-01-01

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of non-local interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα–repeat proteins, which are dominated by local interactions, a combined experimental and simulation analysis was performed on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured sub-millisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine and valine (ILV) side-chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3 and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side-chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely-folded unproductive intermediates are a common property of the βα-repeat motif. PMID:20226790

Thermodynamics and kinetics of protein folding on the ribosome: Alteration in energy landscapes, denatured state, and transition state ensembles

NASA Astrophysics Data System (ADS)

O'Brien, Edward; Vendruscolo, Michele; Dobson, Christopher

2010-03-01

In vitro experiments examining cotranslational folding utilize ribosome-nascent chain complexes (RNCs) in which the nascent chain is stalled at different points of its biosynthesis on the ribosome. We investigate the thermodynamics, kinetics, and structural properties of RNCs containing five different globular and repeat proteins stalled at ten different nascent chain lengths using coarse grained replica exchange simulations. We find that when the proteins are stalled near the ribosome exit tunnel opening they exhibit altered folding coopserativity, quantified by the van't Hoff enthalpy criterion; a significantly altered denatured state ensemble, in terms of Rg and shape parameters (Rg tensor); and the appearance of partially folded intermediates during cotranslation, evidenced by the appearance of a third basin in the free energy profile. These trends are due in part to excluded volume (crowding) interactions between the ribosome and nascent chain. We perform in silico temperature-jump experiments on the RNCs and examine nascent chain folding kinetics and structural changes in the transition state ensemble at various stall lengths.

NMR analysis of a kinetically trapped intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase.

PubMed

Sugimoto, Hayuki; Noda, Yasuo; Segawa, Shin-ichi

2011-09-16

A thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase refolds into a kinetically trapped metastable intermediate when subjected to a rapid lowering of temperature. We attempted to characterise this intermediate using multidimensional NMR spectroscopy. The (1)H-(15)N heteronuclear single quantum coherence spectrum after a rapid temperature decrease (the spectrum of the intermediate) showed good chemical shift dispersion but was significantly different from that of the native state, suggesting that the intermediate adopts a nonnative but well-structured conformation. Large chemical shift changes for the backbone amide protons between the native and the intermediate states were observed for residues in the β-sheet consisting of strands 2, 3, 5, 6, and 7 as well as in the C-terminal region. These residues were found to be in close proximity to aromatic residues, suggesting that the chemical shift changes are mainly due to ring current shifts caused by the aromatic residues. The two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy experiments showed that the intermediate contained substantial, native-like NOE connectivities, although there were fewer cross peaks in the spectrum of the intermediate compared with that of the native state. It was also shown that there were native-like interresidue NOEs for residues buried in the protein, whereas many of the NOE cross peaks were lost for the residues involved in a surface-exposed aromatic cluster. These results suggest that, in the intermediate, the aromatic cluster at the surface is structurally less organised, whereas the interior of the protein has relatively rigid, native-like side-chain packing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

PubMed

Chen, Chao; Cao, Ruikai; Shrestha, Ruben; Ward, Christina; Katz, Benjamin B; Fischer, Christopher J; Tomich, John M; Li, Ping

2015-05-15

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.

Unique Vaginal Microbiota That Includes an Unknown Mycoplasma-Like Organism Is Associated With Trichomonas vaginalis Infection

PubMed Central

Martin, David H.; Zozaya, Marcela; Lillis, Rebecca A.; Myers, Leann; Nsuami, M. Jacques; Ferris, Michael J.

2013-01-01

Background. The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis–infected women differs from that in T. vaginalis–uninfected women. Methods. Vaginal samples from 30 T. vaginalis–infected women were matched by Nugent score to those from 30 T. vaginalis–uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction–amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. Results. Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis–infected and T. vaginalis–uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis–infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. Conclusions. T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response. PMID:23482642

Unique vaginal microbiota that includes an unknown Mycoplasma-like organism is associated with Trichomonas vaginalis infection.

PubMed

Martin, David H; Zozaya, Marcela; Lillis, Rebecca A; Myers, Leann; Nsuami, M Jacques; Ferris, Michael J

2013-06-15

The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis-infected women differs from that in T. vaginalis-uninfected women. Vaginal samples from 30 T. vaginalis-infected women were matched by Nugent score to those from 30 T. vaginalis-uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction-amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis-infected and T. vaginalis-uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis-infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response.

Metabolic profiles of exercise in patients with McArdle disease or mitochondrial myopathy

PubMed Central

Sharma, Rohit; Tadvalkar, Laura; Clish, Clary B.; Haller, Ronald G.; Mootha, Vamsi K.

2017-01-01

McArdle disease and mitochondrial myopathy impair muscle oxidative phosphorylation (OXPHOS) by distinct mechanisms: the former by restricting oxidative substrate availability caused by blocked glycogen breakdown, the latter because of intrinsic respiratory chain defects. We applied metabolic profiling to systematically interrogate these disorders at rest, when muscle symptoms are typically minimal, and with exercise, when symptoms of premature fatigue and potential muscle injury are unmasked. At rest, patients with mitochondrial disease exhibit elevated lactate and reduced uridine; in McArdle disease purine nucleotide metabolites, including xanthine, hypoxanthine, and inosine are elevated. During exercise, glycolytic intermediates, TCA cycle intermediates, and pantothenate expand dramatically in both mitochondrial disease and control subjects. In contrast, in McArdle disease, these metabolites remain unchanged from rest; but urea cycle intermediates are increased, likely attributable to increased ammonia production as a result of exaggerated purine degradation. Our results establish skeletal muscle glycogen as the source of TCA cycle expansion that normally accompanies exercise and imply that impaired TCA cycle flux is a central mechanism of restricted oxidative capacity in this disorder. Finally, we report that resting levels of long-chain triacylglycerols in mitochondrial myopathy correlate with the severity of OXPHOS dysfunction, as indicated by the level of impaired O2 extraction from arterial blood during peak exercise. Our integrated analysis of exercise and metabolism provides unique insights into the biochemical basis of these muscle oxidative defects, with potential implications for their clinical management. PMID:28716914

MAP4 Mechanism that Stabilizes Mitochondrial Permeability Transition in Hypoxia: Microtubule Enhancement and DYNLT1 Interaction with VDAC1

PubMed Central

Zhang, Yi-ming; Zhang, Jia-ping; Hu, Jiong-yu; Zhang, Qiong; Dai, Xia; Teng, Miao; Zhang, Dong-xia; Huang, Yue-sheng

2011-01-01

Mitochondrial membrane permeability has received considerable attention recently because of its key role in apoptosis and necrosis induced by physiological events such as hypoxia. The manner in which mitochondria interact with other molecules to regulate mitochondrial permeability and cell destiny remains elusive. Previously we verified that hypoxia-induced phosphorylation of microtubule-associated protein 4 (MAP4) could lead to microtubules (MTs) disruption. In this study, we established the hypoxic (1% O2) cell models of rat cardiomyocytes, H9c2 and HeLa cells to further test MAP4 function. We demonstrated that increase in the pool of MAP4 could promote the stabilization of MT networks by increasing the synthesis and polymerization of tubulin in hypoxia. Results showed MAP4 overexpression could enhance cell viability and ATP content under hypoxic conditions. Subsequently we employed a yeast two-hybrid system to tag a protein interacting with mitochondria, dynein light chain Tctex-type 1 (DYNLT1), by hVDAC1 bait. We confirmed that DYNLT1 had protein-protein interactions with voltage-dependent anion channel 1 (VDAC1) using co-immunoprecipitation; and immunofluorescence technique showed that DYNLT1 was closely associated with MTs and VDAC1. Furthermore, DYNLT1 interactions with MAP4 were explored using a knockdown technique. We thus propose two possible mechanisms triggered by MAP4: (1) stabilization of MT networks, (2) DYNLT1 modulation, which is connected with VDAC1, and inhibition of hypoxia-induced mitochondrial permeabilization. PMID:22164227

Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems

PubMed Central

Vicens, Alberto; Borziak, Kirill; Karr, Timothy L.; Roldan, Eduardo R.S.

2017-01-01

Abstract Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete. PMID:28333336

Differences in Grain Ultrastructure, Phytochemical and Proteomic Profiles between the Two Contrasting Grain Cd-Accumulation Barley Genotypes

PubMed Central

Sun, Hongyan; Cao, Fangbin; Wang, Nanbo; Zhang, Mian; Mosaddek Ahmed, Imrul; Zhang, Guoping; Wu, Feibo

2013-01-01

To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low- grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars. PMID:24260165

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

PubMed Central

Smit, Judith J; Monteferrario, Davide; Noordermeer, Sylvie M; van Dijk, Willem J; van der Reijden, Bert A; Sixma, Titia K

2012-01-01

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains. PMID:22863777

Identification of Early Response Genes in Human Peripheral Leukocytes Infected with Orientia tsutsugamushi: The Emergent of a Unique Gene Expression Profile for Diagnosis of O. tsutsugamush Infection

DTIC Science & Technology

2010-01-01

dynein to move from the cell periphery to the microtubule organizing center [22]. Therefore, the initial interactions between host and intracellular...used to study host-pathogen interactions , mainly by identifying genes from pathogens that may be involved in pathogenecity and by surveying the scope...toward understanding the host-Orientia tsutsugamushi interaction at the molecular level, we used human cDNA microarray technology to examine in detail

Miro-Working beyond Mitochondria and Microtubules.

PubMed

Tang, Bor Luen

2018-03-04

The small GTPase Miro is best known for its regulation of mitochondrial movement by engaging with the microtubule-based motor proteins kinesin and dynein. Very recent findings have now showed that Miro also targets peroxisomes and regulates microtubule-dependent peroxisome motility. Moreover, Miro recruits and stabilizes the myosin motor Myo19 at the mitochondria to enable actin-based mitochondria movement, which is important for mitochondrial segregation during mitosis. Miro thus has much broader functions that previously known, and these new findings may have important implications on disease pathology.

Genetic variation in fatty acid elongases is not associated with intermediate cardiovascular phenotypes or myocardial infarction

USDA-ARS?s Scientific Manuscript database

Elongases 2, 4 and 5, encoded by genes ELOVL2, ELOVL4 and ELOVL5, have a key role in the biosynthesis of very long chain polyunsaturated fatty acids (PUFAs). To date, few studies have investigated the associations between elongase polymorphisms and cardiovascular health. We investigated whether ELOV...

Hyperammonaemia‐induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress

PubMed Central

Davuluri, Gangarao; Allawy, Allawy; Thapaliya, Samjhana; Rennison, Julie H.; Singh, Dharmvir; Kumar, Avinash; Sandlers, Yana; Van Wagoner, David R.; Flask, Chris A.; Hoppel, Charles; Kasumov, Takhar

2016-01-01

Key points Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia.We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+/NADH ratio and ATP content.During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids.Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell‐permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content.Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. Abstract Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non‐hepatic ammonia disposal. Non‐hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α‐ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+/NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell‐permeable ester of α‐ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia‐induced mitochondrial dysfunction is a novel therapeutic approach. PMID:27558544

Molecular dynamics simulations of polyethers and a quaternary ammonium ionic liquid as CO2 absorbers

NASA Astrophysics Data System (ADS)

Cardoso, Piercarlo Fortunato; Fernandez, Juan S. L. C.; Lepre, Luiz Fernando; Ando, Rômulo Augusto; Costa Gomes, Margarida F.; Siqueira, Leonardo J. A.

2018-04-01

The properties of mixtures of butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, [N4111][NTf2], with poly(ethyleneglycol) dimethyl ether, PEO, were described as a function of PEO chain size by molecular dynamics simulations. Both PEO chain size and mixture composition revealed to play a significant role in determining the structure and the dynamics of the fluids. The remarkably higher viscosity observed for mixtures composed by 0.25 mole fraction of PEO was attributed to the increase in the gauche population of OCCO dihedral of the polyether of longer chains. The negative solvation enthalpy (ΔsolH < 0) and entropy (ΔsolS < 0) revealed a favorable CO2 absorption by the neat and mixture systems. The CO2 absorption was higher in neat PEO, particularly considering longer chains. The gas solubility in the mixtures presented intermediate values in comparison to the neat PEO and neat ionic liquid. The CO2 solutions had their structures discussed in the light of the calculated radial and spatial distribution functions.

Molecular dynamics simulations of polyethers and a quaternary ammonium ionic liquid as CO2 absorbers.

PubMed

Cardoso, Piercarlo Fortunato; Fernandez, Juan S L C; Lepre, Luiz Fernando; Ando, Rômulo Augusto; Costa Gomes, Margarida F; Siqueira, Leonardo J A

2018-04-07

The properties of mixtures of butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, [N 4111 ][NTf 2 ], with poly(ethyleneglycol) dimethyl ether, PEO, were described as a function of PEO chain size by molecular dynamics simulations. Both PEO chain size and mixture composition revealed to play a significant role in determining the structure and the dynamics of the fluids. The remarkably higher viscosity observed for mixtures composed by 0.25 mole fraction of PEO was attributed to the increase in the gauche population of OCCO dihedral of the polyether of longer chains. The negative solvation enthalpy (Δ sol H < 0) and entropy (Δ sol S < 0) revealed a favorable CO 2 absorption by the neat and mixture systems. The CO 2 absorption was higher in neat PEO, particularly considering longer chains. The gas solubility in the mixtures presented intermediate values in comparison to the neat PEO and neat ionic liquid. The CO 2 solutions had their structures discussed in the light of the calculated radial and spatial distribution functions.

Causal Networks or Causal Islands? The Representation of Mechanisms and the Transitivity of Causal Judgment.

PubMed

Johnson, Samuel G B; Ahn, Woo-kyoung

2015-09-01

Knowledge of mechanisms is critical for causal reasoning. We contrasted two possible organizations of causal knowledge—an interconnected causal network, where events are causally connected without any boundaries delineating discrete mechanisms; or a set of disparate mechanisms—causal islands—such that events in different mechanisms are not thought to be related even when they belong to the same causal chain. To distinguish these possibilities, we tested whether people make transitive judgments about causal chains by inferring, given A causes B and B causes C, that A causes C. Specifically, causal chains schematized as one chunk or mechanism in semantic memory (e.g., exercising, becoming thirsty, drinking water) led to transitive causal judgments. On the other hand, chains schematized as multiple chunks (e.g., having sex, becoming pregnant, becoming nauseous) led to intransitive judgments despite strong intermediate links ((Experiments 1-3). Normative accounts of causal intransitivity could not explain these intransitive judgments (Experiments 4 and 5). Copyright © 2015 Cognitive Science Society, Inc.

Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

PubMed

Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2014-05-01

The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands. © 2014 FEBS.

Local spin dynamics at low temperature in the slowly relaxing molecular chain [Dy(hfac)3{NIT(C6H4OPh)}]: A μ+ spin relaxation study

NASA Astrophysics Data System (ADS)

Arosio, Paolo; Corti, Maurizio; Mariani, Manuel; Orsini, Francesco; Bogani, Lapo; Caneschi, Andrea; Lago, Jorge; Lascialfari, Alessandro

2015-05-01

The spin dynamics of the molecular magnetic chain [Dy(hfac)3{NIT(C6H4OPh)}] were investigated by means of the Muon Spin Relaxation (μ+SR) technique. This system consists of a magnetic lattice of alternating Dy(III) ions and radical spins, and exhibits single-chain-magnet behavior. The magnetic properties of [Dy(hfac)3{NIT(C6H4OPh)}] have been studied by measuring the magnetization vs. temperature at different applied magnetic fields (H = 5, 3500, and 16500 Oe) and by performing μ+SR experiments vs. temperature in zero field and in a longitudinal applied magnetic field H = 3500 Oe. The muon asymmetry P(t) was fitted by the sum of three components, two stretched-exponential decays with fast and intermediate relaxation times, and a third slow exponential decay. The temperature dependence of the spin dynamics has been determined by analyzing the muon longitudinal relaxation rate λinterm(T), associated with the intermediate relaxing component. The experimental λinterm(T) data were fitted with a corrected phenomenological Bloembergen-Purcell-Pound law by using a distribution of thermally activated correlation times, which average to τ = τ0 exp(Δ/kBT), corresponding to a distribution of energy barriers Δ. The correlation times can be associated with the spin freezing that occurs when the system condenses in the ground state.

Leptospira species in floodwater during the 2011 floods in the Bangkok Metropolitan Region, Thailand.

PubMed

Thaipadungpanit, Janjira; Wuthiekanun, Vanaporn; Chantratita, Narisara; Yimsamran, Surapon; Amornchai, Premjit; Boonsilp, Siriphan; Maneeboonyang, Wanchai; Tharnpoophasiam, Prapin; Saiprom, Natnaree; Mahakunkijcharoen, Yuvadee; Day, Nicholas P J; Singhasivanon, Pratap; Peacock, Sharon J; Limmathurotsakul, Direk

2013-10-01

Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding.

Chemical Probes for the Functionalization of Polyketide Intermediates**

PubMed Central

Riva, Elena; Wilkening, Ina; Gazzola, Silvia; Li, W M Ariel; Smith, Luke; Leadlay, Peter F; Tosin, Manuela

2014-01-01

A library of functionalized chemical probes capable of reacting with ketosynthase-bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl-ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology. PMID:25212788

On the thermodynamic and kinetic investigations of a [c2]daisy chain polymer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hmadeh, Mohamad; Fang, Lei; Trabolsi, Ali

2010-01-01

We report a variety of [c2]daisy chain molecules which undergo quantitative, efficient, and fully reversible molecular movements upon the addition of base/acid in organic solvents. Such externally triggered molecular movements can induce the contraction and extension of the [c2]daisy chain molecule as a whole. A linear polymer of such a bistable [c2]daisy chain exerts similar types of movements and can be looked upon as a candidate for the development of artificial muscles. The spectrophotometric investigations of both the monomeric and polymeric bistable [c2]daisy chains, as well as the corresponding model compounds, were performed in MeCN at room temperature, in ordermore » to obtain the thermodynamic parameters for these mechanically interlocked molecules. Based on their spectrophotometric and thermodynamic characteristics, kinetic analysis of the acid/base-induced contraction and extension of the [c2]daisy chain monomer and polymer were conducted by employing a stopped-flow technique. These kinetic data suggest that the rates of contraction and extension for these [c2]daisy chain molecules are determined by the thermodynamic stabilities of the corresponding kinetic intermediates. Faster switching rates for both the contraction and extension processes of the polymeric [c2]daisy chain were observed when compared to those of its monomeric counterpart. These kinetic and thermodynamic investigations on [c2]daisy chain-based muscle-like compounds provide important information for those seeking an understanding of the mechanisms of actuation in mechanically interlocked macromolecules.« less

Self assembled linear polymeric chains with tuneable semiflexibility using isotropic interactions.

PubMed

Abraham, Alex; Chatterji, Apratim

2018-04-21

We propose a two-body spherically symmetric (isotropic) potential such that particles interacting by the potential self-assemble into linear semiflexible polymeric chains without branching. By suitable control of the potential parameters, we can control the persistence length of the polymer and can even introduce a controlled number of branches. Thus we show how to achieve effective directional interactions starting from spherically symmetric potentials. The self-assembled polymers have an exponential distribution of chain lengths akin to what is observed for worm-like micellar systems. On increasing particle density, the polymeric chains self-organize to an ordered line-hexagonal phase where every chain is surrounded by six parallel chains, the transition is first order. On further increase in monomer density, the order is destroyed and we get a branched gel-like phase. This potential can be used to model semi-flexible equilibrium polymers with tunable semiflexibility and excluded volume. The use of the potential is computationally cheap and hence can be used to simulate and probe equilibrium polymer dynamics with long chains. The potential also gives a plausible method of tuning colloidal interactions in experiments such that one can obtain self-assembling polymeric chains made up of colloids and probe polymer dynamics using an optical microscope. Furthermore, we show how a modified potential leads to the observation of an intermediate nematic phase of self-assembled chains in between the low density disordered phase and the line-ordered hexagonal phase.

A facile method to synthesize boron-doped Ni/Fe alloy nano-chains as electrocatalyst for water oxidation

NASA Astrophysics Data System (ADS)

Yang, Yisu; Zhuang, Linzhou; Lin, Rijia; Li, Mengran; Xu, Xiaoyong; Rufford, Thomas E.; Zhu, Zhonghua

2017-05-01

We report a novel magnetic field assisted chemical reduction method for the synthesis of boron-doped Ni/Fe nano-chains as promising catalysts for the oxygen evolution reaction (OER). The boron-doped Ni/Fe nano-chains were synthesised in a one step process at room temperature using NaBH4 as a reducing agent. The addition of boron reduced the magnetic moment of the intermediate synthesis products and produced nano-chains with a high specific surface area of 73.4 m2 g-1. The boron-doped Ni/Fe nano-chains exhibited catalytic performance superior to state-of-the-art Ba0.5Sr0.5Co0.8Fe0.2O3-δ perovskite and RuO2 noble metal oxide catalysts. The mass normalized activity of the boron-doped Ni/Fe nano-chains measured at an overpotential of 0.35 V was 64.0 A g-1, with a Tafel slope of only 40 mV dec-1. The excellent performance of the boron-doped Ni/Fe nano-chains can be attributed to the uniform elemental distribution and highly amorphous structure of the B-doped nano-chains. These results provide new insights into the effect of doping transition-metal based OER catalysts with non-metallic elements. The study demonstrates a facile approach to prepare transition metal nano-chains using magnetic field assisted chemical reduction method as cheap and highly active catalysts for electrochemical water oxidation.

Self assembled linear polymeric chains with tuneable semiflexibility using isotropic interactions

NASA Astrophysics Data System (ADS)

Abraham, Alex; Chatterji, Apratim

2018-04-01

We propose a two-body spherically symmetric (isotropic) potential such that particles interacting by the potential self-assemble into linear semiflexible polymeric chains without branching. By suitable control of the potential parameters, we can control the persistence length of the polymer and can even introduce a controlled number of branches. Thus we show how to achieve effective directional interactions starting from spherically symmetric potentials. The self-assembled polymers have an exponential distribution of chain lengths akin to what is observed for worm-like micellar systems. On increasing particle density, the polymeric chains self-organize to an ordered line-hexagonal phase where every chain is surrounded by six parallel chains, the transition is first order. On further increase in monomer density, the order is destroyed and we get a branched gel-like phase. This potential can be used to model semi-flexible equilibrium polymers with tunable semiflexibility and excluded volume. The use of the potential is computationally cheap and hence can be used to simulate and probe equilibrium polymer dynamics with long chains. The potential also gives a plausible method of tuning colloidal interactions in experiments such that one can obtain self-assembling polymeric chains made up of colloids and probe polymer dynamics using an optical microscope. Furthermore, we show how a modified potential leads to the observation of an intermediate nematic phase of self-assembled chains in between the low density disordered phase and the line-ordered hexagonal phase.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

PubMed

Wallmeier, Julia; Shiratori, Hidetaka; Dougherty, Gerard W; Edelbusch, Christine; Hjeij, Rim; Loges, Niki T; Menchen, Tabea; Olbrich, Heike; Pennekamp, Petra; Raidt, Johanna; Werner, Claudius; Minegishi, Katsura; Shinohara, Kyosuke; Asai, Yasuko; Takaoka, Katsuyoshi; Lee, Chanjae; Griese, Matthias; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Sauer, Sascha; Wallingford, John B; Hamada, Hiroshi; Omran, Heymut

2016-08-04

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Genetic analysis of a Y-chromosome region that induces triplosterile phenotypes and is essential for spermatid individualization in Drosophila melanogaster.

PubMed Central

Timakov, B; Zhang, P

2000-01-01

The heterochromatic Y chromosome of Drosophila melanogaster contains approximately 40 Mb of DNA but has only six loci mutable to male sterility. Region h1-h9 on YL, which carries the kl-3 and kl-5 loci, induces male sterility when present in three copies. We show that three separate segments within the region are responsible for the triplosterility and have an additive effect on male fertility. The triplosterile males displayed pleiotropic defects, beginning at early postmeiotic stages. However, the triplosterility was unaffected by kl-3 or kl-5 alleles. These data suggest that region h1-h9 is complex and may contain novel functions in addition to those of the previously identified kl-3 and kl-5 loci. The kl-3 and kl-5 mutations as well as deficiencies within region h1-h9 result in loss of the spermatid axonemal outer dynein arms. Examination using fluorescent probes showed that males deficient for h1-h3 or h4-h9 displayed a postmeiotic lesion with disrupted individualization complexes scattered along the spermatid bundle. In contrast, the kl-3 and kl-5 mutations had no effect on spermatid individualization despite the defect in the axonemes. These results demonstrate that region h1-h9 carries genetically separable functions: one required for spermatid individualization and the other essential for assembling the axonemal dynein arms. PMID:10790393

Testing the time-of-flight model for flagellar length sensing.

PubMed

Ishikawa, Hiroaki; Marshall, Wallace F

2017-11-07

Cilia and flagella are microtubule-based organelles that protrude from the surface of most cells, are important to the sensing of extracellular signals, and make a driving force for fluid flow. Maintenance of flagellar length requires an active transport process known as intraflagellar transport (IFT). Recent studies reveal that the amount of IFT injection negatively correlates with the length of flagella. These observations suggest that a length-dependent feedback regulates IFT. However, it is unknown how cells recognize the length of flagella and control IFT. Several theoretical models try to explain this feedback system. We focused on one of the models, the "time-of-flight" model, which measures the length of flagella on the basis of the travel time of IFT protein in the flagellar compartment. We tested the time-of-flight model using Chlamydomonas dynein mutant cells, which show slower retrograde transport speed. The amount of IFT injection in dynein mutant cells was higher than that in control cells. This observation does not support the prediction of the time-of-flight model and suggests that Chlamydomonas uses another length-control feedback system rather than that described by the time-of-flight model. © 2017 Ishikawa and Marshall. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Two chain gallium fluorodiphosphates: synthesis, structure solution, and their transient presence during the hydrothermal crystallisation of a microporous gallium fluorophosphate.

PubMed

Millange, Franck; Walton, Richard I; Guillou, Nathalie; Loiseau, Thierry; O'Hare, Dermot; Férey, Gérard

2002-04-21

Two novel gallium fluorodiphosphates have been isolated and their structures solved ab initio from powder X-ray diffraction data; the materials readily interconvert under hydrothermal conditions, and are metastable with respect to an open-framework zeolitic gallium fluorophosphate, during the synthesis of which they are present as transient intermediates.

Total synthesis of (+)-antroquinonol and (+)-antroquinonol D.

PubMed

Sulake, Rohidas S; Chen, Chinpiao

2015-03-06

The first total synthesis of (+)-antroquinonol and (+)-antroquinonol D, two structurally unique quinonols with a sesquiterpene side chain, is described. The route features an iridium-catalyzed olefin isomerization-Claisen rearrangement reaction (ICR), lactonization, and Grubbs olefin metathesis. The requisite α,β-unsaturation was achieved via the selenylation/oxidation protocol and elimination of β-methoxy group to provide two natural products from a common intermediate.

Electrostatic forces govern the binding mechanism of intrinsically disordered histone chaperones

PubMed Central

Liu, Chuanbo; Wang, Tianshu; Bai, Yawen; Wang, Jin

2017-01-01

A unified picture to understand the protein recognition and function must include the native binding complex structure ensembles and the underlying binding mechanisms involved in specific biological processes. However, quantifications of both binding complex structures and dynamical mechanisms are still challenging for IDP. In this study, we have investigated the underlying molecular mechanism of the chaperone Chz1 and histone H2A.Z-H2B association by equilibrium and kinetic stopped-flow fluorescence spectroscopy. The dependence of free energy and kinetic rate constant on electrolyte mean activity coefficient and urea concentration are uncovered. Our results indicate a previous unseen binding kinetic intermediate. An initial conformation selection step of Chz1 is also revealed before the formation of this intermediate state. Based on these observations, a mixed mechanism of three steps including both conformation selection and induced fit is proposed. By combination of the ion- and denaturant-induced experiments, we demonstrate that electrostatic forces play a dominant role in the recognition of bipolar charged intrinsically disordered protein Chz1 to its preferred partner H2A.Z-H2B. Both the intra-chain and inter-chain electrostatic interactions have direct impacts on the native collapsed structure and binding mechanism. PMID:28552960

Effects of a Protic Ionic Liquid on the Reaction Pathway during Non-Aqueous Sol–Gel Synthesis of Silica: A Raman Spectroscopic Investigation

PubMed Central

Martinelli, Anna

2014-01-01

The reaction pathway during the formation of silica via a two-component “non-aqueou” sol-gel synthesis is studied by in situ time-resolved Raman spectroscopy. This synthetic route is followed with and without the addition of the protic ionic liquid 1-ethylimidazolium bis(trifluoromethanesulfonyl)imide (C2HImTFSI) in order to investigate its effect on the reaction pathway. We demonstrate that Raman spectroscopy is suitable to discriminate between different silica intermediates, which are produced and consumed at different rates with respect to the point of gelation. We find that half-way to gelation monomers and shorter chains are the most abundant silica species, while the formation of silica rings strongly correlates to the sol-to-gel transition. Thus, curling up of linear chains is here proposed as a plausible mechanism for the formation of small rings. These in turn act as nucleation sites for the condensation of larger rings and thus the formation of the open and polymeric silica network. We find that the protic ionic liquid does not change the reaction pathway per se, but accelerates the cyclization process, intermediated by the faster inclusion of monomeric species. PMID:24743891

Chemical vapor deposition of gallium nitride from the GaCl(3)+NH(3) system. Theoretical study of the structure and thermodynamics of potential intermediates formed in the gaseous phase.

PubMed

Kovács, Attila

2002-06-17

Quantum chemical calculations at the B3P86/6-311G(d,p) level have been performed on potential intermediate molecules in the chemical vapor deposition (CVD) of GaN from the GaCl(3) + NH(3) system. The investigated molecules included the monomer (Cl(x)GaNH(x), x = 1-3) and oligomer species (Cl(2)GaNH(2))(n) with n = 1-3 and (ClGaNH)(n) with n = 1-4 as well as the respective chain dimers and trimers. The calculations revealed the importance of intramolecular Cl...H hydrogen bonding and dipole-dipole interactions in determining the conformational properties of the larger species. Except for the ClGaNH monomer, the Ga[bond]N bonding has a single bond character with a strong ionic contribution. Our thermodynamic study of the composition of the gaseous phase supported the predominance of the Cl(3)GaNH(3) complex under equilibrium conditions. Additionally, the calculated Gibbs free energies of various GaCl(3) + NH(3) reactions imply the favored formation of "saturated" chain and cyclic oligomers below 1000 K.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wujcik, Kevin H.; Wang, Dunyang Rita; Pascal, Tod A.

Lithium sulfur (Li-S) batteries are well known for their high theoretical specific capacities, but are plagued with scientific obstacles that make practical implementation of the technology impossible. The success of Li-S batteries will likely necessitate the use of thick sulfur cathodes that enable high specific energy densities. However, little is known about the fundamental reaction mechanisms and chemical processes that take place in thick cathodes, as most research has focused on studying thinner cathodes that enable high performance. In this study, in situ X-ray absorption spectroscopy at the sulfur K-edge is used to examine the back of a 115 μmmore » thick Li-S cathode during discharge. Our results show that in such systems, where electrochemical reactions between sulfur and lithium are likely to proceed preferentially toward the front of the cathode, lithium polysulfide dianions formed in this region diffuse to the back of the cathode during discharge. We show that high conversion of elemental sulfur is achieved by chemical reactions between elemental sulfur and polysulfide dianions of intermediate chain length (Li 2S x, 4 ≤ x ≤ 6). Our work suggests that controlling the formation and diffusion of intermediate chain length polysulfide dianions is crucial for insuring full utilization of thick sulfur cathodes.« less

Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heroux, A.; Bozinovski, D; Valley, M

2009-01-01

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. Themore » oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.« less

The equilibrium properties and folding kinetics of an all-atom Go model of the Trp-cage.

PubMed

Linhananta, Apichart; Boer, Jesse; MacKay, Ian

2005-03-15

The ultrafast-folding 20-residue Trp-cage protein is quickly becoming a new benchmark for molecular dynamics studies. Already several all-atom simulations have probed its equilibrium and kinetic properties. In this work an all-atom Go model is used to accurately represent the side-chain packing and native atomic contacts of the Trp-cage. The model reproduces the hallmark thermodynamics cooperativity of small proteins. Folding simulations observe that in the fast-folding dominant pathway, partial alpha-helical structure forms before hydrophobic core collapse. In the slow-folding secondary pathway, partial core collapse occurs before helical structure. The slow-folding rate of the secondary pathway is attributed to the loss of side-chain rotational freedom, due to the early core collapse, which impedes the helix formation. A major finding is the observation of a low-temperature kinetic intermediate stabilized by a salt bridge between residues Asp-9 and Arg-16. Similar observations [R. Zhou, Proc. Natl. Acad. Sci. U.S.A. 100, 13280 (2003)] were reported in a recent study using an all-atom model of the Trp-cage in explicit water, in which the salt-bridge stabilized intermediate was hypothesized to be the origin of the ultrafast-folding mechanism. A theoretical mutation that eliminates the Asp-9-Arg-16 salt bridge, but leaves the residues intact, is performed. Folding simulations of the mutant Trp-cage observe a two-state free-energy landscape with no kinetic intermediate and a significant decrease in the folding rate, in support of the hypothesis.

The equilibrium properties and folding kinetics of an all-atom Go xAF model of the Trp-cage

NASA Astrophysics Data System (ADS)

Linhananta, Apichart; Boer, Jesse; MacKay, Ian

2005-03-01

The ultrafast-folding 20-residue Trp-cage protein is quickly becoming a new benchmark for molecular dynamics studies. Already several all-atom simulations have probed its equilibrium and kinetic properties. In this work an all-atom Go ¯ model is used to accurately represent the side-chain packing and native atomic contacts of the Trp-cage. The model reproduces the hallmark thermodynamics cooperativity of small proteins. Folding simulations observe that in the fast-folding dominant pathway, partial α-helical structure forms before hydrophobic core collapse. In the slow-folding secondary pathway, partial core collapse occurs before helical structure. The slow-folding rate of the secondary pathway is attributed to the loss of side-chain rotational freedom, due to the early core collapse, which impedes the helix formation. A major finding is the observation of a low-temperature kinetic intermediate stabilized by a salt bridge between residues Asp-9 and Arg-16. Similar observations [R. Zhou, Proc. Natl. Acad. Sci. U.S.A. 100, 13280 (2003)] were reported in a recent study using an all-atom model of the Trp-cage in explicit water, in which the salt-bridge stabilized intermediate was hypothesized to be the origin of the ultrafast-folding mechanism. A theoretical mutation that eliminates the Asp-9-Arg-16 salt bridge, but leaves the residues intact, is performed. Folding simulations of the mutant Trp-cage observe a two-state free-energy landscape with no kinetic intermediate and a significant decrease in the folding rate, in support of the hypothesis.

AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy.

PubMed

Takahashi, Daisuke; Inomata, Tatsuji; Fukui, Tatsuya

2017-06-26

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Assembly Pathway of Mitochondrial Respiratory Chain Complex I.

PubMed

Guerrero-Castillo, Sergio; Baertling, Fabian; Kownatzki, Daniel; Wessels, Hans J; Arnold, Susanne; Brandt, Ulrich; Nijtmans, Leo

2017-01-10

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain α-ketoacid dehydrogenase

PubMed Central

Naik, Mandar T.; Huang, Tai-Huang

2004-01-01

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain α-ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain α-ketoacids. Recently hbLBD has been found to follow two-step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small β-barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state ↔denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 ± 0.9 K. Cold denaturation of hbLBD is also predicted and discussed. PMID:15322287

Non-conserved magnetization operator and 'fire-and-ice' ground states in the Ising-Heisenberg diamond chain

NASA Astrophysics Data System (ADS)

Torrico, Jordana; Ohanyan, Vadim; Rojas, Onofre

2018-05-01

We consider the diamond chain with S = 1/2 XYZ vertical dimers which interact with the intermediate sites via the interaction of the Ising type. We also suppose all four spins form the diamond-shaped plaquette to have different g-factors. The non-uniform g-factors within the quantum spin dimer as well as the XY-anisotropy of the exchange interaction lead to the non-conserving magnetization for the chain. We analyze the effects of non-conserving magnetization as well as the effects of the appearance of negative g-factors among the spins from the unit cell. A number of unusual frustrated states for ferromagnetic couplings and g-factors with non-uniform signs are found out. These frustrated states generalize the "half-fire-half-ice" state introduced in reference Yin et al. (2015). The corresponding zero-temperature ground state phase diagrams are presented.

Dechlorination of short chain chlorinated paraffins by nanoscale zero-valent iron.

PubMed

Zhang, Zhi-Yong; Lu, Mang; Zhang, Zhong-Zhi; Xiao, Meng; Zhang, Min

2012-12-01

In this study, nanoscale zero-valent iron (NZVI) particles were synthesized and used for the reductive dehalogenation of short chain chlorinated paraffins (SCCPs) in the laboratory. The results show that the dechlorination rate of chlorinated n-decane (CP(10)) by NZVI increased with decreased solution pH. Increasing the loading of NZVI enhanced the dechlorination rate of CP(10). With an increase in temperature, the degradation rate increased. The reduction of CP(10) by NZVI was accelerated with increasing the concentration of humic acid up to 15 mg/L but then was inhibited. The dechlorination of CP(10) within the initial 18 h followed pseudo-first order rate model. The formation of intermediate products indicates a stepwise dechlorination pathway of SCCPs by NZVI. The carbon chain length and chlorination degree of SCCPs have a polynominal impact on dechlorination reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Mechanisms of deterioration of intermediate moisture food systems

NASA Technical Reports Server (NTRS)

Labuza, T. P.

1972-01-01

A study of shelf stability in intermediate moisture foods was made. Major efforts were made to control lipid oxidation and nonenzymatic browning. In order to determine means of preventing these reactions, model systems were developed having the same water activity content relationship of intermediate moisture foods. Models were based on a cellulose-lipid and protein-lipid system with glycerol added as the humectant. Experiments with both systems indicate that lipid oxidation is promoted significantly in the intermediate moisture range. The effect appeared to be related to increased mobility of either reactants or catalysts, since when the amount of water in the system reached a level where capillary condensation occurred and thus free water was present, the rates of oxidation increased. With added glycerol, which is water soluble and thus increases the amount of mobile phase, the increase in oxidation rate occurs at a lower relative humidity. The rates of oxidation were maximized at 61% RH and decreased again at 75% RH probably due to dilution. No significant non-enzymatic browning occurred in the protein-lipid systems. Prevention of oxidation by the use of metal chelating agents was enhanced in the cellulose system, whereas, with protein present, the lipid soluble chain terminating antioxidants (such as BHA) worked equally as well. Preliminary studies of foods adjusted to the intermediate moisture range bear out the results of oxidation in model systems. It can be concluded that for most fat containing intermediate moisture foods, rancidity will be the reaction most limiting stability.

Influence of acid chain length on the properties of TiO2 prepared by sol-gel method and LC-MS studies of methylene blue photodegradation.

PubMed

Bakre, Pratibha V; Volvoikar, Prajesh S; Vernekar, Amit A; Tilve, S G

2016-07-15

Nano-sized titanium dioxide photocatalysts were synthesized by hybrid hydrolytic nonhydrolytic sol-gel method using aliphatic organic acid templates to study the effect of chain length on their properties. X-ray diffraction pattern indicated crystalline anatase phase. The Barrett-Joyner-Halenda surface area measurement gave surface area ranging from 98.4 to 205.5m(2)/g and was found to be dependent on the chain length of the aliphatic acid. The longer chain acids rendered the material with high surface area. The organic acids acted as bidentate ligand and a surfactant in controlling the size and the mesoporosity. The size of the TiO2 nanoparticulate was found to be in the range of 10-18nm. The catalyst prepared by employing long chain acids octanoic acid and palmitic acid had smaller size, narrow pore radius, higher surface area and showed better photocatalytic activity than the commercially available Degussa P25 catalyst for the degradation of methylene blue dye. A new intermediate was identified by tandem liquid chromatography mass spectrometry studies during the degradation of methylene blue solution. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanism of Polyubiquitination by Human Anaphase-Promoting Complex: RING Repurposing for Ubiquitin Chain Assembly

DOE PAGES

Brown, Nicholas G.; Watson, Edmond R.; Weissmann, Florian; ...

2014-10-09

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here in this paper we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms.more » During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.« less

Rouse mode analysis of chain relaxation in polymer nanocomposites

DOE PAGES

Kalathi, Jagannathan T.; Kumar, Sanat K.; Rubinstein, Michael; ...

2015-04-20

Large-scale molecular dynamics simulations are used to study the internal relaxations of chains in nanoparticle (NP)/polymer composites. We examine the Rouse modes of the chains, a quantity that is closest in spirit to the self-intermediate scattering function, typically determined in an (incoherent) inelastic neutron scattering experiment. Our simulations show that for weakly interacting mixtures of NPs and polymers, the effective monomeric relaxation rates are faster than in a neat melt when the NPs are smaller than the entanglement mesh size. In this case, the NPs serve to reduce both the monomeric friction and the entanglements in the polymer melt, asmore » in the case of a polymer–solvent system. However, for NPs larger than half the entanglement mesh size, the effective monomer relaxation is essentially unaffected for low NP concentrations. Even in this case, we observe a strong reduction in chain entanglements for larger NP loadings. Furthermore, the role of NPs is to always reduce the number of entanglements, with this effect only becoming pronounced for small NPs or for high concentrations of large NPs. Our studies of the relaxation of single chains resonate with recent neutron spin echo (NSE) experiments, which deduce a similar entanglement dilution effect.« less

Untangleing the effects of chain rigidity on the structure and dynamics of strongly adsorbed polymer melts

DOE PAGES

Carrillo, Jan-Michael Y.; Cheng, Shiwang; Kumar, Rajeev; ...

2015-06-11

Here, we present a detailed analysis of coarse-grained molecular dynamics simulations of semiflexible polymer melts in contact with a strongly adsorbing substrate. We have characterized the segments in the interfacial layer by counting the number of trains, loops, tails and unadsorbed segments. For more rigid chains, a tail and an adsorbed segment (a train) dominate while loops are more prevalent in more flexible chains. The tails exhibit a non-uniformly stretched conformation akin to the polydispersed pseudobrush envisioned by Guiselin. To probe the dynamics of the segments we computed the layer z-resolved intermediate coherent collective dynamics structure factor, S(q, t, z),more » mean-square displacement of segments, and the 2nd Legendre polynomial of the time-autocorrelation of unit bond vectors, 2[n i(t,z)•n i(0,z)]>. Our results show that segmental dynamics is slower for stiffer chains and there is a strong correlation between the structure and dynamics in the interfacial layer. There is no glassy layer, and the slowing down in dynamics of stiffer chains in the adsorbed region can be attributed to the densification and the more persistent layering of segments.« less

The importance of chain length for the polyphosphate enhancement of acidic potassium permanganate chemiluminescence.

PubMed

Holland, Brendan J; Adcock, Jacqui L; Nesterenko, Pavel N; Peristyy, Anton; Stevenson, Paul G; Barnett, Neil W; Conlan, Xavier A; Francis, Paul S

2014-09-09

Sodium polyphosphate is commonly used to enhance chemiluminescence reactions with acidic potassium permanganate through a dual enhancement mechanism, but commercially available polyphosphates vary greatly in composition. We have examined the influence of polyphosphate composition and concentration on both the dual enhancement mechanism of chemiluminescence intensity and the stability of the reagent under analytically useful conditions. The average chain length (n) provides a convenient characterisation, but materials with similar values can exhibit markedly different distributions of phosphate oligomers. There is a minimum polyphosphate chain length (∼6) required for a large enhancement of the emission intensity, but no further advantage was obtained using polyphosphate materials with much longer average chain lengths. Providing there is a sufficient average chain length, the optimum concentration of polyphosphate is dependent on the analyte and in some cases, may be lower than the quantities previously used in routine detection. However, the concentration of polyphosphate should not be lowered in permanganate reagents that have been partially reduced to form high concentrations of the key manganese(III) co-reactant, as this intermediate needs to be stabilised to prevent formation of insoluble manganese(IV). Copyright © 2014 Elsevier B.V. All rights reserved.

The molecular kink paradigm for rubber elasticity: Numerical simulations of explicit polyisoprene networks at low to moderate tensile strains

NASA Astrophysics Data System (ADS)

Hanson, David E.

2011-08-01

Based on recent molecular dynamics and ab initio simulations of small isoprene molecules, we propose a new ansatz for rubber elasticity. We envision a network chain as a series of independent molecular kinks, each comprised of a small number of backbone units, and the strain as being imposed along the contour of the chain. We treat chain extension in three distinct force regimes: (Ia) near zero strain, where we assume that the chain is extended within a well defined tube, with all of the kinks participating simultaneously as entropic elastic springs, (II) when the chain becomes sensibly straight, giving rise to a purely enthalpic stretching force (until bond rupture occurs) and, (Ib) a linear entropic regime, between regimes Ia and II, in which a force limit is imposed by tube deformation. In this intermediate regime, the molecular kinks are assumed to be gradually straightened until the chain becomes a series of straight segments between entanglements. We assume that there exists a tube deformation tension limit that is inversely proportional to the chain path tortuosity. Here we report the results of numerical simulations of explicit three-dimensional, periodic, polyisoprene networks, using these extension-only force models. At low strain, crosslink nodes are moved affinely, up to an arbitrary node force limit. Above this limit, non-affine motion of the nodes is allowed to relax unbalanced chain forces. Our simulation results are in good agreement with tensile stress vs. strain experiments.

The molecular kink paradigm for rubber elasticity: numerical simulations of explicit polyisoprene networks at low to moderate tensile strains.

PubMed

Hanson, David E

2011-08-07

Based on recent molecular dynamics and ab initio simulations of small isoprene molecules, we propose a new ansatz for rubber elasticity. We envision a network chain as a series of independent molecular kinks, each comprised of a small number of backbone units, and the strain as being imposed along the contour of the chain. We treat chain extension in three distinct force regimes: (Ia) near zero strain, where we assume that the chain is extended within a well defined tube, with all of the kinks participating simultaneously as entropic elastic springs, (II) when the chain becomes sensibly straight, giving rise to a purely enthalpic stretching force (until bond rupture occurs) and, (Ib) a linear entropic regime, between regimes Ia and II, in which a force limit is imposed by tube deformation. In this intermediate regime, the molecular kinks are assumed to be gradually straightened until the chain becomes a series of straight segments between entanglements. We assume that there exists a tube deformation tension limit that is inversely proportional to the chain path tortuosity. Here we report the results of numerical simulations of explicit three-dimensional, periodic, polyisoprene networks, using these extension-only force models. At low strain, crosslink nodes are moved affinely, up to an arbitrary node force limit. Above this limit, non-affine motion of the nodes is allowed to relax unbalanced chain forces. Our simulation results are in good agreement with tensile stress vs. strain experiments.

Polyphosphate kinase: demonstration that short chain polyphosphate serves as a primer for the enzymatic synthesis of polyphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, N.A.; Wood, H.G.

1986-05-01

Polyphosphate (poly(P)) kinase, isolated from Propionibacterium shermanii, catalyzes the following reaction: poly(P/sub n/) + ATPin equilibriumpoly(P/sub n+1/) + ADP. The authors have purified this enzyme to 90% homogeneity and have shown it to be composed of 2-3 identical subunits of M/sub r/ 80,000. Investigation of the reaction mechanism by product analysis has revealed that the elongation phase is processive whereby successive elongation occurs without release of intermediate sizes until very long chains are formed. The initiation phase of synthesis has been investigated using (/sup 32/P) poly(P) primer of chain length 11-60. It is incorporated into long chain poly(P) and themore » /sup 32/P has been shown, by use of poly(P) glucokinase, to be localized at the end of the molecule. Calculation of average chain length based upon the incorporation of /sup 32/P, however, yields a value approx.3 fold higher than the value calculated by another method using poly(P) glucokinase. This result indicates that initiation of poly(P) synthesis occurs by at least one other route which does not involve short chain poly(P) primers. The effect of temperature and concentration of poly(P) primer upon the average chain length of poly(P) synthesized was also investigated. A general trend was observed in which the chain length of the synthesized poly(P) decreased as either temperature or concentration or primer was increased.« less

Mixed Integer Linear Programming model for Crude Palm Oil Supply Chain Planning

NASA Astrophysics Data System (ADS)

Sembiring, Pasukat; Mawengkang, Herman; Sadyadharma, Hendaru; Bu'ulolo, F.; Fajriana

2018-01-01

The production process of crude palm oil (CPO) can be defined as the milling process of raw materials, called fresh fruit bunch (FFB) into end products palm oil. The process usually through a series of steps producing and consuming intermediate products. The CPO milling industry considered in this paper does not have oil palm plantation, therefore the FFB are supplied by several public oil palm plantations. Due to the limited availability of FFB, then it is necessary to choose from which plantations would be appropriate. This paper proposes a mixed integer linear programming model the supply chain integrated problem, which include waste processing. The mathematical programming model is solved using neighborhood search approach.

The primary structure of the hemoglobin of spectacled bear (Tremarctos ornatus, Carnivora).

PubMed

Hofmann, O; Braunitzer, G

1987-08-01

The complete primary structure of the alpha- and beta-chains of the hemoglobin of Spectacled Bear (Tremarctos ornatus) is presented. Following cleavage of the heme-protein link and chain separation by RP-HPLC, their amino-acid sequences were determined by Edman degradation in liquid- and gas-phase sequenators. The hemoglobin of Spectacled Bear displays only five amino-acid exchanges to that of Polar Bear (Ursus maritimus, Ursinae) and Asiatic Black Bear (Ursus tibetanus, Ursinae) whereas 8 and 12 replacements, respectively, to Giant Panda (Ailuropoda melanoleuca) and Lesser Panda (Ailurus fulgens) can be found. This clearly demonstrates that the Spectacled Bear, the most aberrant bear of the Ursidae, is somewhat intermediate between Pandas and Ursinae.

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes.

PubMed

Endrizzi, James A; Beernink, Peter T

2017-11-01

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations. © 2017 The Protein Society.

Functionalized Congeners of P2Y1 Receptor Antagonists:

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Castro, Sonia; Maruoka, Hiroshi; Hong, Kunlun

2010-01-01

The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of anmore » intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e., antithrombotic action.« less

Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals Delta 11- and Delta 9-desaturases with unusual catalytic properties.

PubMed

Liénard, Marjorie A; Lassance, Jean-Marc; Wang, Hong-Lei; Zhao, Cheng-Hua; Piskur, Jure; Johansson, Tomas; Löfstedt, Christer

2010-06-01

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the diversity and complexity of chemical reactions that can be catalysed by pheromone biosynthetic fatty-acyl-CoA desaturases in moths. (c) 2010 Elsevier Ltd. All rights reserved.

Manipulation of double-stranded DNA melting by force

NASA Astrophysics Data System (ADS)

Singh, Amit Raj; Granek, Rony

2017-09-01

By integrating elasticity—as described by the Gaussian network model—with bond binding energies that distinguish between different base-pair identities and stacking configurations, we study the force induced melting of a double-stranded DNA (dsDNA). Our approach is a generalization of our previous study of thermal dsDNA denaturation [J. Chem. Phys. 145, 144101 (2016), 10.1063/1.4964285] to that induced by force at finite temperatures. It allows us to obtain semimicroscopic information about the opening of the chain, such as whether the dsDNA opens from one of the ends or from the interior, forming an internal bubble. We study different types of force manipulation: (i) "end unzipping," with force acting at a single end base pair perpendicular to the helix, (ii) "midunzipping," with force acting at a middle base pair perpendicular to the helix, and (iii) "end shearing," where the force acts at opposite ends along the helix. By monitoring the free-energy landscape and probability distribution of intermediate denaturation states, we show that different dominant intermediate states are stabilized depending on the type of force manipulation used. In particular, the bubble state of the sequence L60B36, which we have previously found to be a stable state during thermal denaturation, is absent for end unzipping and end shearing, whereas very similar bubbles are stabilized by midunzipping, or when the force location is near the middle of the chain. Ours results offer a simple tool for stabilizing bubbles and loops using force manipulations at different temperatures, and may implicate on the mechanism in which DNA enzymes or motors open regions of the chain.

Modulation of frustration in folding by sequence permutation.

PubMed

Nobrega, R Paul; Arora, Karunesh; Kathuria, Sagar V; Graceffa, Rita; Barrea, Raul A; Guo, Liang; Chakravarthy, Srinivas; Bilsel, Osman; Irving, Thomas C; Brooks, Charles L; Matthews, C Robert

2014-07-22

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.

Local spin dynamics at low temperature in the slowly relaxing molecular chain [Dy(hfac)3(NIT(C6H4OPh))]: A μ{sup +} spin relaxation study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arosio, Paolo, E-mail: paolo.arosio@guest.unimi.it; Orsini, Francesco; Corti, Maurizio

2015-05-07

The spin dynamics of the molecular magnetic chain [Dy(hfac){sub 3}(NIT(C{sub 6}H{sub 4}OPh))] were investigated by means of the Muon Spin Relaxation (μ{sup +}SR) technique. This system consists of a magnetic lattice of alternating Dy(III) ions and radical spins, and exhibits single-chain-magnet behavior. The magnetic properties of [Dy(hfac){sub 3}(NIT(C{sub 6}H{sub 4}OPh))] have been studied by measuring the magnetization vs. temperature at different applied magnetic fields (H = 5, 3500, and 16500 Oe) and by performing μ{sup +}SR experiments vs. temperature in zero field and in a longitudinal applied magnetic field H = 3500 Oe. The muon asymmetry P(t) was fitted by the sum of three components, twomore » stretched-exponential decays with fast and intermediate relaxation times, and a third slow exponential decay. The temperature dependence of the spin dynamics has been determined by analyzing the muon longitudinal relaxation rate λ{sub interm}(T), associated with the intermediate relaxing component. The experimental λ{sub interm}(T) data were fitted with a corrected phenomenological Bloembergen-Purcell-Pound law by using a distribution of thermally activated correlation times, which average to τ = τ{sub 0} exp(Δ/k{sub B}T), corresponding to a distribution of energy barriers Δ. The correlation times can be associated with the spin freezing that occurs when the system condenses in the ground state.« less

Use of dynamic simulation to assess the behaviour of linear alkyl benzene sulfonates and their biodegradation intermediates (sulfophenylcarboxylic acids) in estuaries

NASA Astrophysics Data System (ADS)

García-Luque, E.; González-Mazo, E.; Forja, J. M.; Gómez-Parra, A.

2009-02-01

Dynamic laboratory simulation of processes affecting chemical species in their transit through estuaries is a very useful tool to characterize these littoral systems. To date, laboratory studies concerning biodegradation and sorption (onto suspended particulate matter) of LAS in an estuary are scarce. For this reason, a dynamic automated estuarine simulator has been employed to carry out different experiments in order to assess the biodegradability of linear alkyl benzene sulfonates (LAS) and their biodegradation intermediates (sulfophenylcarboxylic acids, SPCs) using environmentally representative LAS concentrations in estuaries by a continuous injection of LAS into the system. During the experiments, a great affinity of LAS for the solid phase has been found, as well as an increased adsorption in line with increased chain length. On the other hand, the presence of SPCs with chain length between 6 and 13 carbon atoms was detected. Accumulation and persistence of medium chain length SPCs (C 6-C 8) along the experiments show that their degradation constitutes the limiting step for the process of LAS mineralization. In the final zone of the simulated estuarine system, the levels of SPCs were below the limits of detection. Thus, the disappearance of SPCs indicated that LAS biodegradation had been completed along the estuary. Similar results have been described for different Iberian littoral ecosystems. Therefore, the simulator employed in this research appears to be a useful tool to anticipate the behaviour of a xenobiotic chemical in its transit through littoral systems with different salinity gradients.

Expected distributions of root-mean-square positional deviations in proteins.

PubMed

Pitera, Jed W

2014-06-19

The atom positional root-mean-square deviation (RMSD) is a standard tool for comparing the similarity of two molecular structures. It is used to characterize the quality of biomolecular simulations, to cluster conformations, and as a reaction coordinate for conformational changes. This work presents an approximate analytic form for the expected distribution of RMSD values for a protein or polymer fluctuating about a stable native structure. The mean and maximum of the expected distribution are independent of chain length for long chains and linearly proportional to the average atom positional root-mean-square fluctuations (RMSF). To approximate the RMSD distribution for random-coil or unfolded ensembles, numerical distributions of RMSD were generated for ensembles of self-avoiding and non-self-avoiding random walks. In both cases, for all reference structures tested for chains more than three monomers long, the distributions have a maximum distant from the origin with a power-law dependence on chain length. The purely entropic nature of this result implies that care must be taken when interpreting stable high-RMSD regions of the free-energy landscape as "intermediates" or well-defined stable states.

Evolution of the biosynthesis of the branched-chain amino acids

NASA Technical Reports Server (NTRS)

Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

1995-01-01

The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

Cellulose microfibril formation within a coarse grained molecular dynamics

NASA Astrophysics Data System (ADS)

Nili, Abdolmadjid; Shklyaev, Oleg; Crespi, Vincent; Zhao, Zhen; Zhong, Linghao; CLSF Collaboration

2014-03-01

Cellulose in biomass is mostly in the form of crystalline microfibrils composed of 18 to 36 parallel chains of polymerized glucose monomers. A single chain is produced by cellular machinery (CesA) located on the preliminary cell wall membrane. Information about the nucleation stage can address important questions about intermediate region between cell wall and the fully formed crystalline microfibrils. Very little is known about the transition from isolated chains to protofibrils up to a full microfibril, in contrast to a large body of studies on both CesA and the final crystalline microfibril. In addition to major experimental challenges in studying this transient regime, the length and time scales of microfibril nucleation are inaccessible to atomistic molecular dynamics. We have developed a novel coarse grained model for cellulose microfibrils which accounts for anisotropic interchain interactions. The model allows us to study nucleation, kinetics, and growth of cellulose chains/protofibrils/microfibrils. This work is supported by the US Department of Energy, Office of Basic Energy Sciences as part of The Center for LignoCellulose Structure and Formation, an Energy Frontier Research Center.

Bacterial Utilization of Ether Glycols

PubMed Central

Fincher, Edward L.; Payne, W. J.

1962-01-01

A soil bacterium capable of using oligo- and polyethylene glycols and ether alcohols as sole sources of carbon for aerobic growth was isolated. The effects of substituent groups added to the ether bonds on the acceptability of the compounds as substrates were studied. Mechanisms for the incorporation of two-carbon compounds were demonstrated by the observation that acetate, glyoxylate, ethylene glycol, and a number of the tricarboxylic acid cycle intermediates served as growth substrates in minimal media. The rate of oxidation of the short-chained ethylene glycols by adapted resting cells varied directly with increasing numbers of two-carbon units in the chains from one to four. The amount of oxygen consumed per carbon atom of oligo- and polyethylene glycols was 100% of theoretical, but only 67% of theoretical for ethylene glycol. Resting cells oxidized oligo- and polyethylene glycols with 2 to 600 two-carbon units in the chains. Longer chained polyethylene glycols (up to 6,000) were oxidized at a very slow rate by these cells. Dehydrogenation of triethylene glycol by adapted cells was observed, coupling the reaction with methylene blue reduction. PMID:13945208

Direct Production of Propene from the Thermolysis of Poly(..beta..-hydroxybutyrate)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mittal, Ashutosh; Pilath, Heidi M.; Johnson, David K.

To transform biomass components into hydrocarbon fuels it is clear that there are two main transformations that need to occur, i.e., deoxygenation and carbon chain extension. The potential routes for decreasing the oxygen content of biomass intermediates include dehydration, hydrodeoxygenation and decarboxylation. One route that is examined here is the conversion of polyhydroxyalkanoates (PHA) to alkenes that would be intermediates to hydrocarbon fuels.Thermal breakdown of PHA proceeds via an intermediate carboxylic acid, which can then be decarboxylated to an alkene. Oligomerization of alkenes by well-known commercial technologies would permit production of a range of hydrocarbon fuels from a carbohydrate derivedmore » intermediate. Moreover, polyhydroxybutyrate (PHB) can be produced in Cupriavidus necator (formerly known as Ralstonia eutropha) and Alcaligenes eutrophus on a variety of carbon sources including glucose, fructose and glycerol with PHB accumulation reaching 75 percent of dry cell mass. We conducted thermal conversion of PHB and pure crotonic acid (CA), the intermediate carboxylic acid produced by thermal depolymerization of PHB, in a flow-through reactor. The results of initial experiments on the thermal conversion of CA showed that up to 75 mole percent yields of propene could be achieved by optimizing the residence time and temperature of the reactor. Further experiments are being investigated to optimize the reactor parameters and enhance propene yields via thermal conversion of PHB.« less

End-growth/evaporation living polymerization kinetics revisited

NASA Astrophysics Data System (ADS)

Semenov, A. N.; Nyrkova, I. A.

2011-03-01

End-growth/evaporation kinetics in living polymer systems with "association-ready" free unimers (no initiator) is considered theoretically. The study is focused on the systems with long chains (typical aggregation number N ≫ 1) at long times. A closed system of continuous equations is derived and is applied to study the kinetics of the chain length distribution (CLD) following a jump of a parameter (T-jump) inducing a change of the equilibrium mean chain length from N0 to N. The continuous approach is asymptotically exact for t ≫ t1, where t1 is the dimer dissociation time. It yields a number of essentially new analytical results concerning the CLD kinetics in some representative regimes. In particular, we obtained the asymptotically exact CLD response (for N ≫ 1) to a weak T-jump (ɛ = N0/N - 1 ≪ 1). For arbitrary T-jumps we found that the longest relaxation time tmax = 1/γ is always quadratic in N (γ is the relaxation rate of the slowest normal mode). More precisely tmax ∝4N2 for N0 < 2N and tmax ∝NN0/(1 - N/N0) for N0 > 2N. The mean chain length Nn is shown to change significantly during the intermediate slow relaxation stage t1 ≪ t ≪ tmax . We predict that N_n(t)-N_n(0)∝ √{t} in the intermediate regime for weak (or moderate) T-jumps. For a deep T-quench inducing strong increase of the equilibrium Nn (N ≫ N0 ≫ 1), the mean chain length follows a similar law, N_n(t)∝ √{t}, while an opposite T-jump (inducing chain shortening, N0 ≫ N ≫ 1) leads to a power-law decrease of Nn: Nn(t)∝t-1/3. It is also shown that a living polymer system gets strongly polydisperse in the latter regime, the maximum polydispersity index r = Nw/Nn being r* ≈ 0.77N0/N ≫ 1. The concentration of free unimers relaxes mainly during the fast process with the characteristic time tf ˜ t1N0/N2. A nonexponential CLD dominated by short chains develops as a result of the fast stage in the case of N0 = 1 and N ≫ 1. The obtained analytical results are supported, in part, by comparison with numerical results found both previously and in the present paper.

Building polyhedra by self-assembly: theory and experiment.

PubMed

Kaplan, Ryan; Klobušický, Joseph; Pandey, Shivendra; Gracias, David H; Menon, Govind

2014-01-01

We investigate the utility of a mathematical framework based on discrete geometry to model biological and synthetic self-assembly. Our primary biological example is the self-assembly of icosahedral viruses; our synthetic example is surface-tension-driven self-folding polyhedra. In both instances, the process of self-assembly is modeled by decomposing the polyhedron into a set of partially formed intermediate states. The set of all intermediates is called the configuration space, pathways of assembly are modeled as paths in the configuration space, and the kinetics and yield of assembly are modeled by rate equations, Markov chains, or cost functions on the configuration space. We review an interesting interplay between biological function and mathematical structure in viruses in light of this framework. We discuss in particular: (i) tiling theory as a coarse-grained description of all-atom models; (ii) the building game-a growth model for the formation of polyhedra; and (iii) the application of these models to the self-assembly of the bacteriophage MS2. We then use a similar framework to model self-folding polyhedra. We use a discrete folding algorithm to compute a configuration space that idealizes surface-tension-driven self-folding and analyze pathways of assembly and dominant intermediates. These computations are then compared with experimental observations of a self-folding dodecahedron with side 300 μm. In both models, despite a combinatorial explosion in the size of the configuration space, a few pathways and intermediates dominate self-assembly. For self-folding polyhedra, the dominant intermediates have fewer degrees of freedom than comparable intermediates, and are thus more rigid. The concentration of assembly pathways on a few intermediates with distinguished geometric properties is biologically and physically important, and suggests deeper mathematical structure.

Low temperature (550-700 K) oxidation pathways of cyclic ketones: Dominance of HO 2-elimination channels yielding conjugated cyclic coproducts

DOE PAGES

Scheer, Adam M.; Welz, Oliver; Vasu, Subith S.; ...

2015-04-13

The low-temperature oxidation of three cyclic ketones, cyclopentanone (CPO; C 5H 8O), cyclohexanone (CHO; C 6H 10 O), and 2-methyl-cyclopentanone (2-Me-CPO; CH 3–C 5H7 O), is studied between 550 and 700 K and at 4 or 8 Torr total pressure. Initial fuel radicals R are formedvia fast H-abstraction from the ketones by laser-photolytically generated chlorine atoms. Intermediates and products from the subsequent reactions of these radicals in the presence of excess O 2 are probed with time and isomeric resolution using multiplexed photoionization mass spectrometry with tunable synchrotron ionizing radiation. For CPO and CHO the dominant product channel in themore » R + O 2 reactions is chain-terminating HO 2-elimination yielding the conjugated cyclic coproducts 2-cyclopentenone and 2-cyclohexenone, respectively. Results on oxidation of 2-Me-CPO also show a dominant contribution from HO 2-elimination. Moreover, the photoionization spectrum of the co-product suggests formation of 2-methyl-2-cyclopentenone and/or 2-cyclohexenone, resulting from a rapid Dowd–Beckwith rearrangement, preceding addition to O 2, of the initial (2-oxocyclopentyl)methyl radical to 3-oxocyclohexyl. Cyclic ethers, markers for hydroperoxyalkyl radicals (QOOH), key intermediates in chain-propagating and chain-branching low-temperature combustion pathways, are only minor products. The interpretation of the experimental results is supported by stationary point calculations on the potential energy surfaces of the associated R + O 2 reactions at the CBS-QB3 level. Furthermore, the calculations indicate that HO 2-elimination channels are energetically favored and product formation via QOOH is disfavored. Lastly, the prominence of chain-terminating pathways linked with HO 2 formation in low-temperature oxidation of cyclic ketones suggests little low-temperature reactivity of these species as fuels in internal combustion engines.« less

Low temperature (550-700 K) oxidation pathways of cyclic ketones: Dominance of HO 2-elimination channels yielding conjugated cyclic coproducts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheer, Adam M.; Welz, Oliver; Vasu, Subith S.

The low-temperature oxidation of three cyclic ketones, cyclopentanone (CPO; C 5H 8O), cyclohexanone (CHO; C 6H 10 O), and 2-methyl-cyclopentanone (2-Me-CPO; CH 3–C 5H7 O), is studied between 550 and 700 K and at 4 or 8 Torr total pressure. Initial fuel radicals R are formedvia fast H-abstraction from the ketones by laser-photolytically generated chlorine atoms. Intermediates and products from the subsequent reactions of these radicals in the presence of excess O 2 are probed with time and isomeric resolution using multiplexed photoionization mass spectrometry with tunable synchrotron ionizing radiation. For CPO and CHO the dominant product channel in themore » R + O 2 reactions is chain-terminating HO 2-elimination yielding the conjugated cyclic coproducts 2-cyclopentenone and 2-cyclohexenone, respectively. Results on oxidation of 2-Me-CPO also show a dominant contribution from HO 2-elimination. Moreover, the photoionization spectrum of the co-product suggests formation of 2-methyl-2-cyclopentenone and/or 2-cyclohexenone, resulting from a rapid Dowd–Beckwith rearrangement, preceding addition to O 2, of the initial (2-oxocyclopentyl)methyl radical to 3-oxocyclohexyl. Cyclic ethers, markers for hydroperoxyalkyl radicals (QOOH), key intermediates in chain-propagating and chain-branching low-temperature combustion pathways, are only minor products. The interpretation of the experimental results is supported by stationary point calculations on the potential energy surfaces of the associated R + O 2 reactions at the CBS-QB3 level. Furthermore, the calculations indicate that HO 2-elimination channels are energetically favored and product formation via QOOH is disfavored. Lastly, the prominence of chain-terminating pathways linked with HO 2 formation in low-temperature oxidation of cyclic ketones suggests little low-temperature reactivity of these species as fuels in internal combustion engines.« less

High doses of S-methylcysteine cause hypoxia-induced cardiomyocyte apoptosis accompanied by engulfment of mitochondaria by nucleus.

PubMed

El-Magd, Mohammed A; Abdo, Walied S; El-Maddaway, Mustafa; Nasr, Nasr M; Gaber, Rasha A; El-Shetry, Eman S; Saleh, Ayman A; Alzahrani, Faisal Abdulrahman Ali; Abdelhady, Doaa H

2017-10-01

Despite its important role as a medicinal plant, some studies reported a toxic effect for garlic (Allium sativum) when given in higher doses. Herein, we investigated the possible cardiotoxic effects of high doses of S-methylcysteine (SMC), a water soluble organosulfur compound present in garlic. Rats were orally administered SMC at a low dose (50mg), high dose (150mg) and very high dose (300mg)/kg body weight, or saline (control) for 10days. High and very high doses of SMC resulted in a significant increase in serum cardiac injury biomarkers [aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin T (cTnT)], as well as oxidative stress marker nitric oxide (NO) concentration in heart and a significant decrease in cardiac superoxide dismutase (SOD) activity. Moreover, ultrastructure findings in myocardium of rats treated by high and very high doses showed inter-bundle vacuolation, loss of myofibrils, and centripetal movement of mitochondria towards nucleus. The mitochondria were partially surrounded by nuclear membrane at high dose SMC, and completely engulfed by nucleus at very high dose. This centripetal movement of mitochondria accompanied by cardiomyocytes hypoxia-induced apoptosis as evident by increasing TUNEL positive cells as well as upregulation of apoptotic genes (caspase3 and Bax), hypoxia inducible factor 1 alpha (HIF1α), dynein light chain 1 (DYNLL1) and downregulation of the anti-apoptotic marker, Bcl2. We conclude that high and very high doses of SMC cause hypoxia induced cardiomyocyte apoptosis accompanied by engulfment of mitochondria by nucleus. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy

NASA Astrophysics Data System (ADS)

Hou, Guangjin; Lu, Xingyu; Vega, Alexander J.; Polenova, Tatyana

2014-09-01

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear 1H-X (X = 13C, 15N, 31P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the 1H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the 1H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from 1H chemical shift anisotropy, while keeping the 1H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [15N]-N-acetyl-valine and [U-13C,15N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate 1H-15N dipolar couplings in the context of 3D experiments is presented on U-13C,15N-enriched dynein light chain protein LC8.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

PubMed

Yoshida, Masa-aki; Yamada, Lixy; Ochi, Hiroe; Iwata, Yoko; Tamura-Nakano, Miwa; Sawada, Hitoshi; Sauer, Warwick H H; Ogura, Atsushi; Hirohashi, Noritaka

2014-08-01

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa. Copyright © 2014 Elsevier Inc. All rights reserved.

The Challenges of Diagnosing Primary Ciliary Dyskinesia

PubMed Central

O'Callaghan, Christopher; Knowles, Michael R.

2011-01-01

Primary ciliary dyskinesia (PCD) is a rare genetic disorder of ciliary structure and function. The diagnosis can be challenging, particularly when using nongenetic assays. The “gold standard” diagnostic test is ultrastructural analysis of respiratory cilia obtained by nasal scrape or brush biopsy. A few specialized centers use high-speed videomicroscopy to examine ciliary beat. Certain beat patterns correlate with ultrastructural defects, and, in some cases, subtle alterations in beat pattern can be seen when ultrastructure is normal. Recent studies have shown that nasal nitric oxide (NO) is very low in patients with PCD compared with healthy control subjects; therefore, this assay may be a useful screening or adjunctive test for PCD. Because acute respiratory illnesses may yield alterations in ciliary ultrastructure, ciliary beat, and nasal NO values, these tests should be performed during a stable baseline period. Identification of an array of PCD genes has provided the opportunity for making a definitive genetic diagnosis for PCD in some cases. All of these approaches have a role in diagnosing PCD. For example, PCD has been confirmed by identifying disease-causing mutations in a heavy dynein chain gene in individuals with normal ciliary ultrastructure but subtle defects in ciliary beat and low nasal NO. Priorities to improve nongenetic diagnostic capability include standardization of nasal NO as a screening test and the development of specialized centers using uniform approaches for the analysis of ciliary ultrastructure and ciliary beat pattern. Another chapter in this issue (see Zariwala and colleagues, pp. 430) addresses the progress toward improved capabilities for definitive genetic testing PMID:21926395

Nitrenes, carbenes, diradicals, and ylides. Interconversions of reactive intermediates.

PubMed

Wentrup, Curt

2011-06-21

Rearrangements of aromatic and heteroaromatic nitrenes and carbenes can be initiated with either heat or light. The thermal reaction is typically induced by flash vacuum thermolysis, with isolation of the products at low temperatures. Photochemical experiments are conducted either under matrix isolation conditions or in solution at ambient temperature. These rearrangements are usually initiated by ring expansion of the nitrene or carbene to a seven-membered ring ketenimine, carbodiimide, or allene (that is, a cycloheptatetraene or an azacycloheptatetraene when a nitrogen is involved). Over the last few years, we have found that two types of ring opening take place as well. Type I is an ylidic ring opening that yields nitrile ylides or diazo compounds as transient intermediates. Type II ring opening produces either dienylnitrenes (for example, from 2-pyridylnitrenes) or 1,7-(1,5)-diradicals (such as those formed from 2-quinoxalinylnitrenes), depending on which of these species is better stabilized by resonance. In this Account, we describe our achievements in elucidating the nature of the ring-opened species and unraveling the connections between the various reactive intermediates. Both of these ring-opening reactions are found, at least in some cases, to dominate the subsequent chemistry. Examples include the formation of ring-opened ketenimines and carbodiimides, as well as the ring contraction reactions that form five-membered ring nitriles (such as 2- and 3-cyanopyrroles from pyridylnitrenes, N-cyanoimidazoles from 2-pyrazinyl and 4-pyrimidinylnitrenes, N-cyanopyrazoles from 2-pyrimidinylnitrenes and 3-pyridazinylnitrenes, and so forth). The mechanisms of formation of the open-chain and ring-contraction products were unknown at the onset of this study. In the course of our investigation, several reactions with three or more consecutive reactive intermediates have been unraveled, such as nitrene, seven-membered cyclic carbodiimide, and open-chain nitrile ylide. It has been possible in some cases to observe them all and determine their interrelationships by means of a combination of matrix-isolation spectroscopy, photochemistry, flash vacuum thermolysis, and computational chemistry. These studies have led to a deeper understanding of the nature of reactive intermediates and chemical reactivity. Moreover, the results indicate new directions for further exploration: ring-opening reactions of carbenes, nitrenes, and cyclic cumulenes can be expected in many other systems.

Application of small-angle neutron scattering to the study of forces between magnetically chained monodisperse ferrofluid emulsion droplets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jain, Dr Nirmesh; Liu, Dr C K; Hawkett, Dr B. S.

2014-01-01

The optical magnetic chaining technique (MCT) developed by Leal-Calderon, Bibette and co-workers in the 1990 s allows precise measurements of force profiles between droplets in monodisperse ferrofluid emulsions. However, the method lacks an in-situ determination of droplet size and therefore requires the combination of separately acquired measurements of droplet chain periodicity versus an applied magnetic field from optical Bragg scattering and droplet diameter inferred from dynamic light scattering (DLS) to recover surface force-distance profiles between the colloidal particles. Compound refractive lens (CRL) focussed small-angle scattering (SANS) MCT should result in more consistent measurements of droplet size (form factor measurements inmore » the absence of field) and droplet chaining period (from structure factor peaks when the magnetic field is applied); and, with access to shorter length scales, extend force measurements to closer approaches than possible by optical measurements. We report on CRL-SANS measurements of monodisperse ferrofluid emulsion droplets aligned in straight chains by an applied field perpendicular to the incident beam direction. Analysis of the scattering from the closely spaced droplets required algorithms that carefully treated resolution and its effect on mean scattering vector magnitudes in order to determine droplet size and chain periods to sufficient accuracy. At lower applied fields scattering patterns indicate structural correlations transverse to the magnetic field direction due to the formation of intermediate structures in early chain growth.« less

Entanglement spreading after a geometric quench in quantum spin chains

NASA Astrophysics Data System (ADS)

Alba, Vincenzo; Heidrich-Meisner, Fabian

2014-08-01

We investigate the entanglement spreading in the anisotropic spin-1/2 Heisenberg (XXZ) chain after a geometric quench. This corresponds to a sudden change of the geometry of the chain or, in the equivalent language of interacting fermions confined in a box trap, to a sudden increase of the trap size. The entanglement dynamics after the quench is associated with the ballistic propagation of a magnetization wave front. At the free fermion point (XX chain), the von Neumann entropy SA exhibits several intriguing dynamical regimes. Specifically, at short times a logarithmic increase is observed, similar to local quenches. This is accurately described by an analytic formula that we derive from heuristic arguments. At intermediate times partial revivals of the short-time dynamics are superposed with a power-law increase SA˜tα, with α <1. Finally, at very long times a steady state develops with constant entanglement entropy, apart from oscillations. As expected, since the model is integrable, we find that the steady state is nonthermal, although it exhibits extensive entanglement entropy. We also investigate the entanglement dynamics after the quench from a finite to the infinite chain (sudden expansion). While at long times the entanglement vanishes, we demonstrate that its relaxation dynamics exhibits a number of scaling properties. Finally, we discuss the short-time entanglement dynamics in the XXZ chain in the gapless phase. The same formula that describes the time dependence for the XX chain remains valid in the whole gapless phase.

In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors*

PubMed Central

Fort, Alfredo G.; Murray, John W.; Dandachi, Nadine; Davidson, Michael W.; Dermietzel, Rolf; Wolkoff, Allan W.; Spray, David C.

2011-01-01

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μm of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μm ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4–0.5 μm/s, which was inhibited with 1 mm of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μm vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32. PMID:21536677

In vivo control mechanisms of motor-cargo movement on microtubules

NASA Astrophysics Data System (ADS)

Gunawardena, Shermali

2014-03-01

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. Previous work suggested that the amyloid precursor protein (APP) functions as a kinesin-1 receptor during transport. However, how APP vesicle motility is regulated is unclear. Using genetics and in vivo imaging in Drosophila we showed that reduction of presenilin (PS) substantially increased anterograde and retrograde APP vesicle velocities. Strikingly, PS deficiency had no effect on an unrelated cargo vesicle containing synaptotagmin, which is powered by a different kinesin motor. Increased PS-mediated velocities required functional kinesin-1 and dynein motors. We also found that these PS-mediated effects on motor protein function were mediated via a pathway that involves glycogen synthase kinase-3 β (GSK-3 β) . PS genetically interacted with GSK-3 β in an activity dependent manner. Excess of active GSK-3 β perturbed transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein, while excess of non-functional GSK-3 β had no effect. Strikingly, GSK-3 β-activity dependent transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3 β are required for normal motor protein function, and we propose a model in which PS likely regulates GSK-3 β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during vesicle motility on microtubules.

Concise CIO based precession-nutation formulations

NASA Astrophysics Data System (ADS)

Capitaine, N.; Wallace, P. T.

2008-01-01

Context: The IAU 2000/2006 precession-nutation models have precision goals measured in microarcseconds. To reach this level of performance has required series containing terms at over 1300 frequencies and involving several thousand amplitude coefficients. There are many astronomical applications for which such precision is not required and the associated heavy computations are wasteful. This justifies developing smaller models that achieve adequate precision with greatly reduced computing costs. Aims: We discuss strategies for developing simplified IAU 2000/2006 precession-nutation procedures that offer a range of compromises between accuracy and computing costs. Methods: The chain of transformations linking celestial and terrestrial coordinates comprises frame bias, precession-nutation, Earth rotation and polar motion. We address the bias and precession-nutation (NPB) portion of the chain, linking the Geocentric Celestial Reference System (GCRS) with the Celestial Intermediate Reference System (CIRS), the latter based on the Celestial Intermediate Pole (CIP) and Celestial Intermediate Origin (CIO). Starting from direct series that deliver the CIP coordinates X,Y and (via the quantity s + XY/2) the CIO locator s, we look at the opportunities for simplification. Results: The biggest reductions come from truncating the series, but some additional gains can be made in the areas of the matrix formulation, the expressions for the nutation arguments and by subsuming long period effects into the bias quantities. Three example models are demonstrated that approximate the IAU 2000/2006 CIP to accuracies of 1 mas, 16 mas and 0.4 arcsec throughout 1995-2050 but with computation costs reduced by 1, 2 and 3 orders of magnitude compared with the full model. Appendices A to G are only available in electronic form at http://www.aanda.org

Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways.

PubMed

Aghera, Nilesh; Udgaonkar, Jayant B

2012-07-13

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Metabolism of Tetralin in Fischer 344 Rats

DTIC Science & Technology

1986-04-01

evaluated petroleum and shale-derived JP-5, a jet fuel composed of aliphatic and aromatic hydrocarbons with the majority of the straight-chain...much like gasoline. JP-8 is a mixture of hydrocarbons of intermediate boiling point and volatility and is similar to the civilian jet fuel , A-1. DFM...toxicity of conventional versus shale-derived JP-5 jet fuel : Light microscopy, hematologic, and serum chemistry studies. Toxicol Appl Pharmacol, 57

Detecting and Correcting Scale Drift in Test Equating: An Illustration from a Large Scale Testing Program

ERIC Educational Resources Information Center

Puhan, Gautam

2009-01-01

The purpose of this study is to determine the extent of scale drift on a test that employs cut scores. It was essential to examine scale drift for this testing program because new forms in this testing program are often put on scale through a series of intermediate equatings (known as equating chains). This process may cause equating error to…

Scale Drift in Equating on a Test That Employs Cut Scores. Research Report. ETS RR-07-34

ERIC Educational Resources Information Center

Puhan, Gautam

2007-01-01

The purpose of this study is to determine the extent of scale drift on a test that employs cut scores. It is essential to examine scale drift in a testing program using new forms that are often put on scale through a series of intermediate equatings (known as equating chains). This may cause equating error to accumulate to a point where scale…

Financial crisis, virtual carbon in global value chains, and the importance of linkage effects. The Spain-china case.

PubMed

López, Luis-Antonio; Arce, Guadalupe; Zafrilla, Jorge

2014-01-01

Trade has a disproportionate environmental impact, while the international fragmentation of production promotes different patterns of intermediate inputs and final goods. Therefore, we split up the balance of domestic embodied emissions in trade (BDEET) to assess it. We find that Spain has a significant emissions deficit with China between 2005 and 2011. The Global Financial Crisis of 2008 reduced Spanish imports of pollution-intensive inputs from China and slightly improved the BDEET. China primarily exports indirect virtual carbon, representing 86% of the total, especially from Production of electricity, gas, and water sector. These linkages effects in China indicate that post-Kyoto agreements must focus not only on traded goods but also on the environmental efficiency of all domestic production chains. The methodology proposed allows us to identify the agents responsible for this trade in both Spain and China, namely the sectors importing intermediate inputs (Construction and Transport equipment) and industries and consumers importing final goods (Textiles, Other manufactures, Computers, and Machinery). The relevant sectors uncertainties found when we compare the results for BDEET and emissions embodied in bilateral trade (BEET) lead us to recommend the former methodology to evaluate the implications of environmental and energy policy for different industries and agents.

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

PubMed

Bedgood, R M; Stallcup, M R

1992-04-05

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.

Acetic Acid Can Catalyze Succinimide Formation from Aspartic Acid Residues by a Concerted Bond Reorganization Mechanism: A Computational Study

PubMed Central

Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

2015-01-01

Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism. PMID:25588215

Acetic acid can catalyze succinimide formation from aspartic acid residues by a concerted bond reorganization mechanism: a computational study.

PubMed

Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

2015-01-12

Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

Hepatocyte Transplantation Improves Phenotype and Extends Survival in a Murine Model of Intermediate Maple Syrup Urine Disease

PubMed Central

Skvorak, Kristen J; Paul, Harbhajan S; Dorko, Kenneth; Marongiu, Fabio; Ellis, Ewa; Chace, Donald; Ferguson, Carolyn; Gibson, K Michael; Homanics, Gregg E; Strom, Stephen C

2009-01-01

Maple syrup urine disease (MSUD; OMIM 248600) is an inborn error of metabolism of the branched chain α-ketoacid dehydrogenase (BCKDH) complex that is treated primarily by dietary manipulation of branched-chain amino acids (BCAA). Dietary restriction is lifelong and compliance is difficult. Liver transplantation significantly improves outcomes; however, alternative therapies are needed. To test novel therapies such as hepatocyte transplantation (HTx), we previously created a murine model of intermediate MSUD (iMSUD), which closely mimics human iMSUD. LacZ-positive murine donor hepatocytes were harvested and directly injected (105 cells/50 µl) into liver of iMSUD mice (two injections at 1–10 days of age). Donor hepatocytes engrafted into iMSUD recipient liver, increased liver BCKDH activity, improved blood total BCAA/alanine ratio, increased body weight at weaning, and extended the lifespan of HTx-treated iMSUD mice compared to phosphate-buffered saline (PBS)–treated and untreated iMSUD mice. Based on these data demonstrating partial metabolic correction of iMSUD in a murine model, coupled to the fact that multiple transplants are possible to enhance these results, we suggest that HTx represents a promising therapeutic intervention for MSUD that warrants further investigation. PMID:19436271

Chemical degradation and morphological instabilities during focused ion beam prototyping of polymers.

PubMed

Orthacker, A; Schmied, R; Chernev, B; Fröch, J E; Winkler, R; Hobisch, J; Trimmel, G; Plank, H

2014-01-28

Focused ion beam processing of low melting materials, such as polymers or biological samples, often leads to chemical and morphological instabilities which prevent the straight-forward application of this versatile direct-write structuring method. In this study the behaviour of different polymer classes under ion beam exposure is investigated using different patterning parameters and strategies with the aim of (i) correlating local temperatures with the polymers' chemistry and its morphological consequences; and (ii) finding a way of processing sensitive polymers with lowest chemical degradation while maintaining structuring times. It is found that during processing of polymers three temperature regimes can be observed: (1) at low temperatures all polymers investigated show stable chemical and morphological behaviour; (2) very high temperatures lead to strong chemical degradation which entails unpredictable morphologies; and (3) in the intermediate temperature regime the behaviour is found to be strongly material dependent. A detailed look reveals that polymers which rather cross-link in the proximity of the beam show stable morphologies in this intermediate regime, while polymers that rather undergo chain scission show tendencies to develop a creeping phase, where material follows the ion beam movement leading to instable and unpredictable morphologies. Finally a simple, alternative patterning strategy is suggested, which allows stable processing conditions with lowest chemical damage even for challenging polymers undergoing chain scission.

A new approach to the side chain formation of 24-alkyl-22-hydroxy steroids: application to the preparation of early brassinolide biosynthetic precursors.

PubMed

Hurski, Alaksiej L; Zhabinskii, Vladimir N; Khripach, Vladimir A

2012-06-01

A new synthetic route to 22S-hydroxy-24R-methyl steroids has been developed and applied for the preparation of cathasterone, (22S)-hydroxycampesterol, and 6-deoxocathasterone, which are precursors in the early stages of the biosynthesis of brassinolide. The construction of the steroid side chain with the correct stereochemistry at C-24 is based on the use of Claisen rearrangement. The introduction of the 22-hydroxyl group has been achieved by epoxidation of the Δ(22)-double bond, nucleophilic opening of the intermediate mesyl epoxide with sodium sulfide, and desulfurization of the formed tetrahydrothiophenes with Raney nickel. Copyright © 2012 Elsevier Inc. All rights reserved.

Linear alkane polymerization on a gold surface.

PubMed

Zhong, Dingyong; Franke, Jörn-Holger; Podiyanachari, Santhosh Kumar; Blömker, Tobias; Zhang, Haiming; Kehr, Gerald; Erker, Gerhard; Fuchs, Harald; Chi, Lifeng

2011-10-14

In contrast to the many methods of selectively coupling olefins, few protocols catenate saturated hydrocarbons in a predictable manner. We report here the highly selective carbon-hydrogen (C-H) activation and subsequent dehydrogenative C-C coupling reaction of long-chain (>C(20)) linear alkanes on an anisotropic gold(110) surface, which undergoes an appropriate reconstruction by adsorption of the molecules and subsequent mild annealing, resulting in nanometer-sized channels (1.22 nanometers in width). Owing to the orientational constraint of the reactant molecules in these one-dimensional channels, the reaction takes place exclusively at specific sites (terminal CH(3) or penultimate CH(2) groups) in the chains at intermediate temperatures (420 to 470 kelvin) and selects for aliphatic over aromatic C-H activation.

Nanoscale Particle Motion in Attractive Polymer Nanocomposites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senses, Erkan; Narayanan, Suresh; Mao, Yimin

Using x-ray photon correlation spectroscopy, we examined slow nanoscale motion of silica nanoparticles individually dispersed in entangled poly (ethylene oxide) melt at particle volume fractions up to 42 %. The nanoparticles, therefore, serve as both fillers for the resulting attractive polymer nanocomposites and probes for the network dynamics therein. The results show that the particle relaxation closely follows the mechanical reinforcement in the nanocomposites only at the intermediate concentrations below the critical value for the chain confinement. Quite unexpectedly, the relaxation time of the particles does not further slowdown at higher volume fractions- when all chains are practically on themore » nanoparticle interface- and decouples from the elastic modulus of the nanocomposites that further increases orders of magnitude.« less

Nanoscale Particle Motion in Attractive Polymer Nanocomposites

DOE PAGES

Senses, Erkan; Narayanan, Suresh; Mao, Yimin; ...

2017-12-06

Using x-ray photon correlation spectroscopy, we examined slow nanoscale motion of silica nanoparticles individually dispersed in entangled poly (ethylene oxide) melt at particle volume fractions up to 42 %. The nanoparticles, therefore, serve as both fillers for the resulting attractive polymer nanocomposites and probes for the network dynamics therein. The results show that the particle relaxation closely follows the mechanical reinforcement in the nanocomposites only at the intermediate concentrations below the critical value for the chain confinement. Quite unexpectedly, the relaxation time of the particles does not further slowdown at higher volume fractions- when all chains are practically on themore » nanoparticle interface- and decouples from the elastic modulus of the nanocomposites that further increases orders of magnitude.« less

Transitions of tethered chain molecules under tension.

PubMed

Luettmer-Strathmann, Jutta; Binder, Kurt

2014-09-21

An applied tension force changes the equilibrium conformations of a polymer chain tethered to a planar substrate and thus affects the adsorption transition as well as the coil-globule and crystallization transitions. Conversely, solvent quality and surface attraction are reflected in equilibrium force-extension curves that can be measured in experiments. To investigate these effects theoretically, we study tethered chains under tension with Wang-Landau simulations of a bond-fluctuation lattice model. Applying our model to pulling experiments on biological molecules we obtain a good description of experimental data in the intermediate force range, where universal features dominate and finite size effects are small. For tethered chains in poor solvent, we observe the predicted two-phase coexistence at transitions from the globule to stretched conformations and also discover direct transitions from crystalline to stretched conformations. A phase portrait for finite chains constructed by evaluating the density of states for a broad range of solvent conditions and tensions shows how increasing tension leads to a disappearance of the globular phase. For chains in good solvents tethered to hard and attractive surfaces we find the predicted scaling with the chain length in the low-force regime and show that our results are well described by an analytical, independent-bond approximation for the bond-fluctuation model for the highest tensions. Finally, for a hard or slightly attractive surface the stretching of a tethered chain is a conformational change that does not correspond to a phase transition. However, when the surface attraction is sufficient to adsorb a chain it will undergo a desorption transition at a critical value of the applied force. Our results for force-induced desorption show the transition to be discontinuous with partially desorbed conformations in the coexistence region.

Alkaloids in the human food chain--natural occurrence and possible adverse effects.

PubMed

Koleva, Irina I; van Beek, Teris A; Soffers, Ans E M F; Dusemund, Birgit; Rietjens, Ivonne M C M

2012-01-01

Alkaloid-containing plants are an intrinsic part of the regular Western diet. The present paper summarizes the occurrence of alkaloids in the food chain, their mode of action and possible adverse effects including a safety assessment. Pyrrolizidine alkaloids are a reason for concern because of their bioactivation to reactive alkylating intermediates. Several quinolizidine alkaloids, β-carboline alkaloids, ergot alkaloids and steroid alkaloids are active without bioactivation and mostly act as neurotoxins. Regulatory agencies are aware of the risks and have taken or are considering appropriate regulatory actions for most alkaloids. These vary from setting limits for the presence of a compound in feed, foods and beverages, trying to define safe upper limits, advising on a strategy aiming at restrictions in use, informing the public to be cautious or taking specific plant varieties from the market. For some alkaloids known to be present in the modern food chain, e.g., piperine, nicotine, theobromine, theophylline and tropane alkaloids risks coming from the human food chain are considered to be low if not negligible. Remarkably, for many alkaloids that are known constituents of the modern food chain and of possible concern, tolerable daily intake values have so far not been defined. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.